Method for increasing resistance against stress factors in plants

ABSTRACT

The invention relates to methods for generating or increasing the resistance, in plants, to at least one biotic or abiotic stress factor, preferably to plant pathogens, by increasing the expression of at least one Bax inhibitor 1 (BI1) protein in at least one plant tissue, with the proviso that the expression in the leaf epidermis remains essentially unchanged. The invention furthermore relates to recombinant expression cassettes and vectors which comprise a nucleic acid sequence coding for a BI protein under the control of a tissue-specific promoter, the promoter having essentially no activity in the leaf epidermis. The invention furthermore relates to recombinant plants transformed with said expression cassettes or vectors, to cultures, parts or recombinant propagation material derived from these plants, and to the use of same for the production of foodstuffs, feeding stuffs, seed, pharmaceuticals or fine chemicals.

The invention relates to methods for generating or increasing theresistance, in plants, to at least one biotic or abiotic stress factor,preferably to plant pathogens, by increasing the expression of at leastone Bax inhibitor 1 (BI1) protein in at least one plant tissue, with theproviso that the expression in the leaf epidermis remains essentiallyunchanged. The invention furthermore relates to recombinant expressioncassettes and vectors which comprise a nucleic acid sequence coding fora BI protein under the control of a tissue-specific promoter, thepromoter having essentially no activity in the leaf epidermis. Theinvention furthermore relates to recombinant plants transformed withsaid expression cassettes or vectors, to cultures, parts or recombinantpropagation material derived from these plants, and to the use of samefor the production of foodstuffs, feeding stuffs, seed, pharmaceuticalsor fine chemicals.

The aim of plant biotechnology work is the generation of plants withadvantageous novel properties, for example for increasing agriculturalproductivity, increasing the quality in the case of foodstuffs, or forproducing specific chemicals or pharmaceuticals. The plant's naturaldefense mechanisms against pathogens are frequently insufficient. Fungaldiseases alone result in annual yield losses of many billions of US$.The introduction of foreign genes from plants, animals or microbialsources can increase the defenses. Examples are the protection oftobacco against feeding damage by insects by expressing Bacillusthuringiensis endotoxins (Vaeck et al. (1987) Nature 328:33-37) or byprotecting tobacco against fungal disease by expressing a bean chitinase(Broglie et al. (1991) Science 254: 1194-1197). However, most of theapproaches described only offer resistance to a single pathogen or anarrow spectrum of pathogens.

Only a few approaches exist which confer, to plants, a resistance to abroader spectrum of pathogens, in particular fungal pathogens. Systemicacquired resistance (SAR)— a defense mechanism in a variety ofplant/pathogen interactions—can be conferred by application ofendogenous messenger substances such as jasmonic acid (JA) or salicylicacid (SA) (Ward et al. (1991) Plant Cell 3:1085-1094; Uknes et al.(1992) Plant Cell 4(6):645-656). Similar effects can also be achieved bysynthetic compounds such as 2,6-dichloroisonicotinic acid (DCINA) orS-methyl benzo[1,2,3)thiadiazol-7-thiocarboxylate (BTH; Bion®)(Friedrich et al. (1996) Plant J 10(1):61-70; Lawton et al. (1996) PlantJ 10:71-82). The expression of the pathogenesis-related (PR) proteins,which are upregulated in the case of SAR, may also cause pathogenresistance in some cases.

In barley, the Mlo locus is described as a negative regulator ofpathogen defense. The loss or loss of function, of the Mlo gene causesan increased, race-unspecific resistance against a large number ofmildew isolates (Büschges R et al. (1997) Cell 88:695-705; Jorgensen J H(1977) Euphytica 26:55-62; Lyngkjaer M F et al. (1995) Plant Pathol44:786-790).

The Mlo gene is described (Büschges R et al. (1997) Cell 88:695-705; WO98/04586; Schulze-Lefert P, Vogel J (2000) Trends Plant Sci. 5:343-348).Various Mlo homologs from other cereal species have been isolated.Methods using these genes for obtaining pathogen resistance have beendescribed (WO 98/04586; WO 00/01722; WO 99/47552). The disadvantage isthat Mlo-deficient plants also initiate the abovementioned defensemechanisms in the absence of a pathogen, which manifests itself in aspontaneous dying of leaf cells (Wolter M et al. (1993) Mol Gen Genet239:122-128). This is why mlo-resistant plants suffer a yield loss ofapproximately 5% (Jörgensen J H (1992) Euphytica 63: 141-152).Furthermore, the spontaneous dying of the leaf cells brings about adisadvantageous hypersusceptibility to necrotrophic and hemibiotrophicpathogens such as Magnaporte grisea (M. grisea) or Cochliobolus sativus(Bipolaris sorokiniana) (Jarosch B et al. (1999) Mol Plant MicrobeInteract 12:508-514; Kumar J et al. (2001) Phytopathology 91:127-133).

Factors which mediate an effect against necrotrophic fungi which can becompared with the mlo resistance have not been identified to date. Thereason for this may be the specific infection mechanism of thenecrotrophic fungi: instead of an appressoria-mediated penetration, theyfirst release mycotoxins and enzymes into the plant host cell, whichleads to the death of the cell. Only then is the cell penetrated(Shirasu K and Schulze-Lefert P (2000) Plant Mol Biol 44:371-385).Similar infection strategies are employed by bacterial pathogens such asErwinia carotovora (Whitehead N A et al. (2002) Antonie van Leeuwenhoek81: 223-231). Penetration resistance with the aid of the formation ofpapillae is no efficient defense strategy in such a case.

Apoptosis, also referred to as programmed cell death, is an essentialmechanism for maintaining tissue homeostasis, and thus constitutes anegatively regulating mechanism which counteracts cell division. Inmulticelled organisms, apoptosis is a natural part of ontogenesis andinvolves, inter alia, the development of the organs and the removal ofsenescent, infected or mutated cells. Apoptosis allows efficientelimination of undesired cells to take place. Interference with, orinhibition of, apoptosis contributes to the pathogenesis of a range ofdiseases, inter alia carcinogenesis. The main effectors of apoptosis areaspartate-specific cysteine proteases, known as caspases. They can beactivated by means of at least two apoptotic signal pathways: firstly,the activation of the TNF (tumor necrosis factor) receptor family;secondly, the central role played by mitochondria. The activation of themitochondrial apoptotic signal pathway is regulated by proteins of theBcl-2 family. This protein family consists of antiapoptotic andproapoptotic proteins such as, for example, Bax. In the case of anapoptotic stimulus, the Bax protein undergoes an allostericconformational change, which leads to the protein being anchored in themitochondrial external membrane and in its oligomerization. The resultof these oligomers is that proapoptotic molecules are released from themitochondria into the zytosol, and these molecules bring about anapoptotic signal cascade and, eventually, the degradation of specificcellular substrates, which results in the death of the cell. The Baxinhibitor 1 BI1 has been isolated via its property to inhibit theproapoptotic effect of BAX (Xu Q & Reed J C (1998) Mol Cell 1(3):337-346). BI1 is a highly conserved protein. It is predominantly foundas integral constituent of intracellular membranes. BI1 interacts withbcl-2 and bcl-x1. The overexpression of BI1 in mammalian cellssuppresses the proapoptotic effect of BAX, etoposide and staurosporin,but not of Fas antigen (Roth W and Reed J C (2002) Nat Med 8: 216-218).In contrast, the inhibition of BI1 by antisense RNA induces apoptosis(Xu Q & Reed J C (1998) Mol Cell 1(3):337-346). The first plant homologsof BI1 have been isolated from rice and Arabidopsis (Kawai et al. (1999)FEBS Lett 464:143-147; Sanchez et al (2000) Plant J 21:393-399). Theseplant proteins suppress the BAX-induced cell death in yeast. The aminoacid sequence homology with human BI1 amounts to approximately 45%. TheArabidopsis homolog AtBI1 is capable of suppressing, in recombinantplants, the pro-apoptotic effect of murine BAX (Kawai-Yamada et al.(2001) Proc Natl Acad Sci USA 98(21):12295-12300). The rice (Oryzasativa) BI1 homolog OsBI1 is expressed in all plant tissues (Kawai etal. (1999) FEBS Lett 464: 143-147). Furthermore described are BI1 genesfrom barley (Hordeum vulgare; GenBank Acc. No.: AJ290421), rice (GenBankAcc. No.: AB025926), Arabidopsis (GenBank Acc. No.: AB025927), tobacco(GenBank Acc. No.: AF390556) and oilseed rape (GenBank Acc. No.:AF390555, Bolduc N et al. (2003) Planta 216:377-386). The expression ofBI1 in barley is upregulated as the result of infection with mildew(Hückelhoven R et al. (2001) Plant Mol Biol 47(6):739-748).

WO 00/26391 describes the overexpression of the anti-apoptotic genesCed-9 from C. elegans, sfIAP from Spodoptera frugiperda, bcl-2 fromhuman and bcl-x1 from chicken in plants for increasing the resistance tonecrotrophic or hemibiotrophic fungi. Plant BI1 homologs are notdisclosed. The expression is under the control of constitutivepromoters. Furthermore described is the expression of a BI1 protein fromArabidopsis under the strong constitutive 35S CaMV promoter in ricecells, and a resistance, induced thereby, to cell-death-inducingsubstances from Magnaporthe grisea (Matsumura H et al. (2003) Plant J33:425-434).

Surprisingly, it has been found within the scope of the presentinvention that, while constitutive expression of a BI1 protein bringsabout resistance to necrotrophic fungi, the result is the breaking ofthe mlo-mediated resistance to the obligate-biotrophic Powdery Mildew(see comparative experiment 1). This questions the economical use of themethods described in the prior art.

The object was to provide plant pathogen defense methods which makepossible an efficient defense against plant pathogens (preferablynecrotrophic pathogens) without breaking any other existing resistanceto other pathogens (such as, for example, biotrophic pathogens). Thisobject is achieved by the method according to the invention.

A first subject of the invention relates to methods for generating orincreasing the resistance, in plants, to at least one biotic or abioticstress factor, comprising the following steps:

-   a) increasing the amount of protein, or the function, of at least    one Bax inhibitor-1 (BI1) protein in at least one plant tissue with    the proviso that the expression in the leaf epidermis remains    essentially unchanged or is reduced, and-   b) selection of the plants in which, in comparison with the starting    plant, a resistance to at least one biotic or abiotic stress factor    exists or is increased.

The biotic or abiotic stress factor is preferably a pathogen, especiallypreferably a pathogen selected from the group of the necrotrophic andhemibiotrophic pathogens.

By epidermis, the skilled worker means the predominant epidermal tissueof primary aerial plant parts, for example of the shoot, the leaves,flowers, fruits and seeds. The epidermal cells secrete outwardly awater-repellent layer, the cuticle. The roots are surrounded by therhizodermis, which, in many ways, resembles the epidermis, but alsoshows pronounced differences. While the outermost layer of the apicalmeristem gives rise to the epidermis, the formation of the rhizodermisis much less clear. Depending on the species, it can be considered, inphylogenetic terms, either as part of the calyptra or as part of theprimary cortex. The epidermis has a number of functions: it protects theplant against desiccation and regulates the transpiration rate. Itprotects the plant against a wide range of chemical and physicalexternal influences, against being fed upon by animals and againstattack by parasites. It is involved in gas exchange, in the secretion ofcertain metabolites and in the absorption of water. It comprisesreceptors for light and mechanical stimuli. It thus acts as signaltransformer between the environment and the plant. In accordance withits various functions, the epidermis comprises a number of differentlydifferentiated cells. To this must be added species-specific variantsand different organizations of the epidermides in the individual partsof a plant. Essentially, it consists of three categories of cells: the“actual” epidermal cells, the cells of the stomata and of the trichomes(Greek: Trichoma, hair), epidermal appendages of varying shape,structure and function. The “actual”, i.e. the least specializedepidermal cells, account for most of the bulk of the cells of theepidermal tissue. In topview, they appear either polygonal (slab orplate shaped) or elongated. The walls between them are often wavy orsinuate. It is not known what induces this shape during development;existing hypotheses only offer unsatisfactory explanations herefor.Elongated epidermal cells can be found in organs or parts of organs thatare elongated themselves, thus, for example, in stems, petioles, leafveins and on the leaves of most monocots. The upper surface andundersurface of laminae can be covered in epidermides with differentstructures, it being possible for the shape of the cells, the wallthickness and the distribution and number of specialized cells (stomataand/or trichomes) per unit area to vary. A high degree of variation isalso found within individual families, for example in the Crassulaceae.In most cases, the epidermis consists of a single layer, thoughmulti-layered water-storing epidermides have been found among speciesfrom a plurality of families (Moraceae: most Ficus species; Piperaceae:Peperonia, Begoniaceae, Malvaceae and the like). Epidermal cells howeversecrete a cuticle on the outside which covers all epidermal surfaces asan uninterrupted film. It may either be smooth or structured by bulges,rods, folds and furrows. However, the folding of the cuticle, which canbe observed when viewing the surface, is not always caused by cuticularrods. Indeed, there are cases where cuticular folding is merely theexpression of the underlying bulges of the cell wall. Epidermalappendages of various form, structure and function are referred to astrichomes and, in the present context, likewise come under the term“epidermis”. They occur in the form of protective hairs, supportivehairs and gland hairs in the form of scales, different papillae and, inthe case of roots, as absorbent hairs. They are formed exclusively byepidermal cells. Frequently, a trichome is formed by only one such acell, however, occasionally, more than one cell is involved in itsformation.

The term “epidermis” likewise comprises papillae. Papillae are bulges ofthe epidermal surface. The textbook example are the papillae on flowersurfaces of pansy (Viola tricolor) and the leaf surfaces of many speciesfrom tropical rain forests. They impart a velvet-like consistency to thesurface. Some epidermal cells can form water stores. A typical exampleare the water vesicles at the surfaces of many Mesembryanthemum speciesand other succulents. In some plants, for example in the case ofcampanula (Campanula persicifolia), the outer walls of the epidermis arethickened like a lens.

The main biomass of all tissues is the parenchyma. The parenchymatictissues include the mesophyll which, in leaves, can be differentiatedinto palisade parenchyma and spongy parenchyma.

Accordingly the skilled worker understands, by mesophyll, aparenchymatic tissue. Parenchymatic cells are always alive, in mostcases isodiametric, rarely elongated. The pith of the shoots, thestorage tissues of the fruits, seeds, the root and other undergroundorgans are also parenchymas, as is the mesophyll.

In the leaves of most ferns and phanerogams, especially in the case ofthe dicotts and many monocotts, the mesophyll is subdivided intopalisade parenchymas and spongy parenchymas. A “typical” leaf is ofdorsiventral organization. In most cases, the palisade parenchyma is atthe upper surface of the leaf immediately underneath the epidermis. Thesponge parenchyma fills the underlying space. It is interspersed by avoluminous intercellular system whose gas space is in direct contactwith the external space via the stomata.

The palisade parenchyma consists of elongated cylindrical cells. In somespecies, the cells are irregular, occasionally bifurcate (Y-shaped: armpalisade parenchyma). Such variants are found in ferns, conifers and afew angiosperms (for example in some Ranunculaceae and Caprifoliaceaespecies [example: elder]). Besides the widest-spread organization formwhich has just been described, the following variants have been found:palisade parenchyma at the leaf undersurface. Particularly conspicuouslyin scaly leaves. (for example arbor vitae (thuja), and in the leaves ofwild garlic (Allium ursinum). Palisade parenchyma at both leaf surfaces(upper surface and undersurface). Frequently found in plants of dryhabitats (xerophytes). Example: prickly lettuce (Lactuca serriola);ring-shaped closed palisade parenchyma: in cylindrically organizedleaves and in conifers' needles.

The variability of the cells of the spongy parenchyma, and theorganization of the spongy parenchyma itself, are even more varied thanthat of the palisade parenchyma. It is most frequently referred to asaerenchyma since it comprises a multiplicity of interconnectedintercellular spaces. The mesophyll may comprise what is known as theassimilation tissue, but the terms mesophyll and assimilation tissue arenot to be used synonymously. There are chloroplast-free leaves whoseorganization differs only to a minor extent from comparable greenleaves. As a consequence, they comprise mesophyll, but assimilation doesnot take place; conversely, assimilation also takes place in, forexample, sections of the shoot. Further aids for characterizingepidermis and mesophyll can be found by the skilled worker for examplein v. GUTTENBERG, H.: Lehrbuch der Allgemeinen Botanik [Textbook ofgeneral botany]. Berlin: Akademie-Verlag 1955 (5th Ed.), HABERLANDT, G.:Physiologische Pflanzenanatomie [Physiological plant anatomy]. Leipzig:W. Engelmann 1924 (6th Ed.); TROLL, W.: Morphologie der Pflanzen [Plantmorphology]. Volume 1: Vegetationsorgane [Vegetation organs]. Berlin:Gebr. Borntraeger, 1937; TROLL, W.: Praktische Einführung in diePflanzenmorphologie [Practical introduction to plant morphology]. Jena:VEB G. Thieme Verlag 1954/1957; TROLL, W., HÖHN, K.: Allgemeine Botanik[General botany]. Stuttgart: F. Enke Verlag, 1973 (4th Ed.)

In one embodiment, the epidermis is characterized in biochemical terms.In one embodiment, the epidermis can be characterized by the activity ofone or more of the following promoters:

-   -   WIR5 (=GstA1), acc. X56012, Dudler & Schweizer, unpublished.    -   GLP4, acc. AJ310534; Wei, Y.; Zhang, Z.; Andersen, C. H.;        Schmelzer, E.; Gregersen, P. L.; Collinge, D. B.;        Smedegaard-Petersen, V.; Thordal-Christensen, H. (1998) An        epidermis/-papilla-specific oxalate oxidase-like protein in the        defence response of barley attacked by the powdery mildew        fungus. Plant Molecular Biology 36, 101-112.    -   GLP2a, acc. AJ237942, Schweizer, P., Christoffel, A. and        Dudler, R. (1999). Transient expression of members of the        germin-like gene family in epidermal cells of wheat confers        disease resistance, Plant J 20, 541-552.    -   Prx7, acc. AJ003141, Kristensen B K, Ammitzböll H, Rasmussen S K        & Nielsen K A. 2001. Transient expression of a vacuolar        peroxidase increases susceptibility of epidermal barley cells to        powdery mildew. Molecular Plant Pathology, 2(6), 311-317    -   GerA, acc. AF250933; Wu S, Druka A, Horvath H, Kleinhofs A,        Kannangara G & von Wettstein D, 2000. Functional        characterization of seed coat-specific members of the barley        germin gene family. Plant Phys Biochem 38, 685-698    -   OsROC1, acc. AP004656    -   RTBV, acc. AAV62708, AAV62707; Klöti, A, Henrich C, Bieri S, He        X, Chen G, Burkhardt P K, Wünn J, Lucca, P, Hohn, T, Potrykus I        & Fütterer J, 1999, Upstream and downstream sequence elements        determine the specificity of the rice tungro bacilliform virus        promoter and influence RNA production after transcription        initiation. PMB 40, 249-266

In one embodiment, the epidermis comprises the fact that all theabovementioned promoters are active in the tissue or the cell. Inanother embodiment, the epidermis comprises the fact that only some ofthe promoters are active, for example 2, 3, 5 or 7 or more, but at leastfrom only one of those detailed above.

In one embodiment, the mesophyll is characterized in biochemical terms.In one embodiment, the mesophyll can be characterized by the activity ofone or more of the following promoters:

-   -   PPCZm1 (=PEPC); Kausch, A. P., Owen, T. P., Zachwieja, S. J.,        Flynn, A. R. and Sheen, J. (2001) Mesophyll-specific, light and        metabolic regulation of the C(4)PPCZm1 promoter in transgenic        maize. Plant Mol. Biol. 45, 1-15    -   OsrbcS, Kyozuka et al PlaNT Phys: 1993 102: Kyozuka J, McElroy        D, Hayakawa T, Xie Y, Wu R & Shimamoto K. 1993. Light-regulated        and cell-specific expression of tomato rbcs-gusA and rice        rbcs-gusA fusion genes in transgenic rice. Plant Phys 102,        991-1000    -   OsPPDK, acc. AC099041, unpublished.    -   TaGF-2.8, acc. M63223; Schweizer, P., Christoffel, A. and        Dudler, R. (1999). Transient expression of members of the        germin-like gene family in epidermal cells of wheat confers        disease resistance, Plant J 20, 541-552.    -   TaFBPase, acc. X53957 unpublished.    -   TaWIS1, acc. AF467542; U.S. 200220115849    -   HvBIS1, acc. AF467539; U.S. 200220115849    -   ZmMIS1, acc. AF467514; U.S. 200220115849    -   HvPR1a, acc. X74939; Bryngelsson et al. Molecular Plant-Microbe        Interactions (1994)    -   HvPR1b, acc. X74940; Bryngelsson et al. Molecular Plant-Microbe        Interactions (1994)    -   HvB1, 3gluc; acc. AF479647; unpublished.    -   HvPrx8, acc. AJ276227; Kristensen et al MPP 2001 (see above)    -   HvPAL, acc. X97313; Wei, Y.; Zhang, Z.; Andersen, C. H.;        Schmelzer, E.; Gregersen, P. L.; Collinge, D. B.;        Smedegaard-Petersen, V.; Thordal-Christensen, H. (1998) An        epidermis/-papilla-specific oxalate oxidase-like protein in the        defence response of barley attacked by the powdery mildew        fungus. Plant Molecular Biology 36, 101-112.

In one embodiment, the mesophyll comprises the fact that all theabovementioned promoters are active in the tissue or the cell. Inanother embodiment, the mesophyll comprises the fact that only some ofthe promoters are active, for example 2, 3, 5 or 7 or more, but at leastfrom only one of those detailed above.

In one embodiment, all of the abovementioned promoters are active in aplant used or produced in accordance with the invention or in theepidermis and in the mesophyll in a plant according to the invention. Inone embodiment, only some of the abovementioned promoters are active,for example 2, 5, 7 or more; however, at least one of the promotersdetailed above is active in each case.

In a preferred embodiment, the increase in the protein quantity orfunction of the BI1 protein takes place in a root-, tuber- ormesophyll-specific manner, especially preferably in a mesophyll-specificmanner, for example by recombinant expression of a nucleic acid sequencecoding for said BI1 protein under the control of a root-, tuber- ormesophyll-specific promoter, preferably under the control of amesophyll-specific promoter.

As described in the present text, in one embodiment, the expression orfunction, in the mesophyll of a plant, of the protein according to theinvention or of the BI-1 characterized in the present text is increased.An increase in expression can be achieved as described hereinbelow. Byincreased expression or function, the present text means both theactivation or enhancement of the expression or function of theendogenous protein including a de novo expression, but also an increasein or enhancement as the result of the expression of a transgenicprotein or factor.

In an especially preferred embodiment, the increase in the proteinquantity or function of at least one plant BI1 protein can be combinedwith an mlo-resistant phenotype or with the inhibition or reduction, incomparison with a control plant, of the expression of MLO, RacB and/orNaOx in the plant or a part thereof, for example in a tissue, butespecially advantageously at least in the epidermis or a considerablenumber of the epidermal cells and/or with the increase in the expressionor function of PEN2 and/or PEN1 in the plant, for exampleconstitutively, or a part thereof, for example in a tissue, butespecially advantageously at least in the epidermis or in a considerablenumber of the epidermal cells, with the proviso that the expression of aplant BI1 protein in the leaf epidermis remains essentially unchanged oris reduced, thus providing a combined resistance to both necrotrophy andbiotrophic pathogens.

The Mlo locus has been described in barley as negative regulator ofpathogen defense. The loss, or loss of function, of the Mlo gene bringsabout an increased, race-unspecific resistance to a number of mildewisolates (Büschges R et al. (1997) Cell 88:695-705; Jorgensen J H (1977)Euphytica 26:55-62; Lyngkjaer M F et al. (1995) Plant Pathol44:786-790).

The Mlo gene has been described (Büschges R et al. (1997) Cell88:695-705; WO 98/04586; Schulze-Lefert P, Vogel J (2000) Trends PlantSci. 5:343-348). Various Mlo homologs from other cereal species havebeen isolated.

An mlo-resistant phenotype can be obtained as described in the priorart. Methods using these genes for obtaining a pathogen resistance aredescribed, inter alia, in WO 98/04586; WO 00/01722; WO 99/47552.

In one embodiment of the present invention, the activity, expression orfunction of MLO, RacB and/or NaOx in the plant or a part thereof, forexample in a tissue, but especially advantageously at least in theepidermis or a substantial number of epidermal cells can advantageouslybe inhibited or reduced in comparison with a control plant or a partthereof. By reducing the activity or function of MLO, RacB and/or NaOxin the plant or a part thereof, for example in a tissue, but especiallyadvantageously at least in the epidermis or a substantial number ofepidermal cells, it is preferred to increase the resistance, orwithstanding power, to biotrophic pathogens in plants produced inaccordance with the invention. This is especially advantageous incombination with a reduction or suppression of cell death due tonecrosis. The activity or function of MLO, RacB and/or NaOx can bereduced or inhibited analogously to what has been described for MLO inWO 98/04586; WO 00/01722; WO 99/47552 and the other publicationsmentioned hereinbelow, whose content is herewith expressly incorporatedinto the present description, in particular for describing the activityand inhibition of MLO. The description of the abovementionedpublications describes processes, methods and especially preferredembodiments for reducing or inhibiting the activity or function of MLO;the examples detail specifically how this can be performed.

The reduction of the activity or function, if appropriate theexpression, of RacB is described in detail in WO 2003020939, which isherewith expressly incorporated into the present description. Thedescription of the abovementioned publication describes processes andmethods for reducing or inhibiting the activity or function of BI-1; theexamples detail specifically how this can be performed. It is especiallypreferred to carry out the reduction or inhibition of the activity orfunction of RacB as described in the embodiments and the examples whichare especially preferred in WO 2003020939 and in the organisms specifiedtherein as being especially preferred, in particular in a plant or apart thereof, for example in a tissue, but especially advantageously atleast in the epidermis or a substantial number of epidermal cells. Thereduction of the activity or function, if appropriate the expression, ofRacB is described in detail in WO 2003020939. In WO 2003020939, theskilled worker can find the sequences which code for RacB proteins andcan also identify RacB by means of the method provided in WO 2003020939.

The reduction of the activity or function, if appropriate of theexpression, of NaOX is described in detail in PCT/EP/03/07589 which isherewith expressly incorporated into the present description. Thedescription of the abovementioned publication describes processes andmethods for reducing or inhibiting the activity or function of NaOx; theexamples detail specifically how this can be performed. It is especiallypreferred to carry out the reduction or inhibition of the activity orfunction of NaOx as described in the embodiments and the examples whichare especially preferred in PCT/EP/03/07589 and in the organismsspecified therein as being especially preferred, in particular in aplant or a part thereof, for example in a tissue, but especiallyadvantageously at least in the epidermis or a substantial number ofepidermal cells. In PCT/EP/03/07589, the skilled worker can find thesequences which code for NaOx proteins and can also identify NaOx bymeans of the method provided in PCT/EP/03/07589.

In one embodiment of the present invention, the activity, expression orfunction of PEN1, PEN2 and/or SNAP34 can advantageously be increased inthe plant, for example constitutively, or in a part thereof, for examplein a tissue, but especially advantageously at least in the epidermis ora substantial number of epidermal cells. The increase in activity, whichalso comprises a de novo expression, of PEN1, PEN2 and/or SNAP 34 in theplant, for example constitutively, or in a part thereof, for example ina tissue, but especially advantageously at least in the epidermis or asubstantial number of epidermal cells will preferably increase theresistance or withstanding power to biotrophic pathogens in the plantsproduced in accordance with the invention. This is especiallyadvantageous in combination with a reduction or suppression of celldeath due to necrosis. The increase in the activity or function, ifappropriate the expression, of PEN2 is described in detail inWO03074688, which is herewith expressly incorporated into the presentdescription. The description of the abovementioned publicationsdescribes processes and methods for reducing or inhibiting the activityor function of PEN2; the examples detail specifically how this can beperformed. The reduction or inhibition of the activity or function ofPEN2 is especially preferably carried out in accordance with theembodiments and examples which are especially preferred in WO03074688and in the organisms detailed therein as being especially preferred, inparticular in plants, for example constitutively, or in a part thereof,for example in a tissue, but especially advantageously at least in theepidermis or a considerable part of the epidermal cells. In WO03074688,the skilled worker will find the sequences which code for PEN2 proteinsand can also identify PEN2 by means of the method provided inWO03074688.

The expression of PEN1 and SNAP34 can be increased analogously to themethods described in WO03074688. Owing to his general expert knowledgeand the prior art with which he is familiar, the skilled worker canisolate and overexpress PEN1 and SNAP34 nucleic acid sequences andprotein sequences. SEQ ID NO: 39 describes the nucleic acid sequencewhich codes for PEN1 from barley; the protein sequence is described inSEQ ID No: 40.

SEQ ID NO: 41 describes the nucleic acid sequence which codes for PEN1from Arabidopsis thaliana; the protein sequence is described in SEQ IDNO: 42. PEN1 from Arabidopsis thaliana is published under the accessionnumbers NM 202559 and NM 112015. The homolog from barley is disclosed inaccession numbers AY246907 and AY246906 as ROR2. They are members of thefairly large family of the syntaxin proteins. Thus, the skilled workercan use simple homology comparisons for identifying further syntaxinproteins which are expressed as potential resistance genes in the methodaccording to the invention.

SEQ ID NO: 43 describes the nucleic acid sequence which codes for SNAP34from barley; the protein sequence is described in SEQ ID NO: 44. TheSNAP-34 homolog from barley is also published as AY 247208 (SNAP-34).Homologs whose function is unknown and which might play a role in theresistance are published as AY 247209 (SNAP-28) and AY 247210 (SNAP-25).The following Arabidopsis genes show a higher degree of homology withbarley SNAP34 than barley SNAP-28 or SNAP-25 to SNAP-34 and can thusadvantageously be co-overexpressed as potential resistance-mediatinggenes:

-   AAM 62553-Arabidopsis SNAP25a-   NP 200929-Arabidopsis SNAP33b-   NP 172842-Arabidopsis SNAP30-   NP 196405-Arabidopsis SNAP29

Accordingly, the invention also relates to a plant in which apolypeptide which is encoded by a nucleic acid molecule comprising thesequences shown in SEQ. ID NO: 39, 41 or 43 or one of the sequencesshown in the abovementioned database publications or which comprises oneof the amino acid sequences shown in the abovementioned databasepublications or in SEQ. ID No.: 40, 42 or 44, or which is a functionalequivalent thereof or which has at least 50%, preferably 70%, morepreferably 80%, even more preferably 90% or more homology with theabovementioned sequences at the coding nucleic acid molecule level or,preferably, at the amino acid level is overexpressed at leastfurthermore in the epidermis, or relates to a plant in which theabove-characterized polypeptide is activated, or its activity orfunction increased, constitutively or in a part, for example in atissue, but especially advantageously at least in the epidermis or asubstantial number of epidermal cells.

A reduction of the expression or activity can be brought about by themethods with which the skilled worker is familiar, for examplemutagenesis, for example EMS, if appropriate TILLING, iRNA; ribozyme,silencing, knockout, and the like. Reduction methods are described inparticular in WO 2003020939, whose methods can readily be adapted to thesequences described herein, which is why the content of WO 2003020939 isexplicitly incorporated herein.

The lowering or reduction of the expression of a BI-1 protein, the BI-1activity or the BI-1 function can be performed in many ways.

“Lowering”, “to lower”, “reduction” or “to reduce” is to be understoodin the broad sense in connection with a BI-1 protein, a BI-1 activity orBI-1 function and comprises the partial or essentially completeprevention or blocking of the functionality of a BI-1 protein, as theresult of different cell-biological mechanisms.

A reduction for the purposes of the invention also comprises aquantitative reduction of a BI-1 protein down to an essentially completeabsence of the BI-1 protein (i.e. lacking detectability of BI-1 activityor BI-1 function or lacking immunological detectability of the BI-1protein). In this context, the expression of a certain BI-1 protein orthe BI-1 activity, or BI-1 function, in a cell or an organism ispreferably reduced by more than 50%, especially preferably by more than80%, very especially preferably by more than 90%.

The invention comprises a variety of strategies for reducing theexpression of a BI-1 protein, the BI-1 activity or the BI-1 function.The skilled worker will recognize that a series of different methods isavailable for influencing the expression of a BI-1 protein, the BI-1activity or the BI-1 function in the desired manner.

A reduction of the BI-1 activity or the BI-1 function is preferablyachieved by reduced expression of an endogenous BI-1 protein.

A reduction of the amount of BI-1 protein, the BI-1 activity or the BI-1function can be effected using the following methods:

-   a) introduction of a double-stranded BI-1 RNA nucleic acid sequence    (BI-1-dsRNA), or of an expression cassette(s) which ensure(s) the    expression thereof;-   b) introduction of a BI-1 antisense nucleic acid sequence, or of an    expression cassette which ensures the expression thereof. Comprised    are those methods in which the antisense nucleic acid sequence is    directed against a BI-1 gene (that is, genomic DNA sequences) or    against a BI-1 gene transcript (that is, RNA sequences). Also    comprised are α-anomeric nucleic acid sequences;-   c) Introduction of a BI-1 antisense nucleic acid sequence in    combination with a ribozyme, or of an expression cassette which    ensures the expression thereof;-   d) Introduction of BI-1 sense nucleic acid sequences for inducing    cosuppression, or of an expression cassette which ensures the    expression thereof;-   e) Introduction of a nucleic acid sequence coding for    dominant-negative BI-1 protein, or of an expression cassette which    ensures the expression thereof;-   f) Introduction of DNA- or protein-binding factors against BI-1    genes, BI-1 RNAs or BI-1 proteins, or of an expression cassette    which ensures their expression;-   g) Introduction of viral nucleic acid sequences and expression    constructs which bring about degradation of the BI-1 RNA, or of an    expression cassette which ensures the expression thereof;-   h) Introduction of constructs for inducing homologous recombination    at endogenous BI-1 genes, for example for the generation of knockout    mutants;-   i) Introduction of mutations in endogenous BI-1 genes for generating    a loss of function (for example generation of stop codons,    reading-frame shifts and the like),    it being necessary for each of the abovementioned methods to be    carried out in an epidermis-specific manner, i.e. the expression in    the epidermal tissue remains unchanged or is reduced. In this    context, each of these methods can bring about a reduction of the    BI-1 expression, BI-1 activity or BI-1 function as defined in the    invention. A combined use is also feasible. Further methods are    known to the skilled worker and may comprise hindering or preventing    the processing of the BI-1 protein, of the transport of the BI-1    protein or its mRNA, inhibition of ribosomal attachment, inhibition    of RNA splicing, induction of a BI-1-RNA-degrading enzyme and/or    inhibition of the elongation or termination of translation.

The epidermis-specific reduction can be effected for example by thetransient application of the abovementioned methods to epidermal cellsor by a specific transformation of essentially only epidermal cells orby the expressional control of the abovementioned constructs under anepidermis-specific promoter or other epidermis-specific control element.

The individual methods which are preferred shall now be describedbriefly in the following text:

-   a) Introduction of a double-stranded BI-1 RNA nucleic acid molecule    (BI-1-dsRNA)

The method of regulating genes by means of double-stranded RNA(“double-stranded RNA interference”; dsRNAi) has been describedrepeatedly in animal and plant organisms (for example Matzke M A et al.(2000) Plant Mol Biol 43:401-415; Fire A. et al (1998) Nature391:806-811; WO 99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO00/44895; WO 00/49035; WO 00/63364). The processes and methods describedin the above references are expressly referred to. Efficient genesuppression can also be demonstrated in the case of transient expressionor following transient transformation, for example as the result of abiolistic transformation (Schweizer P et al. (2000) Plant J 2000 24:895-903). dsRNAi methods are based on the phenomenon that thesimultaneous introduction of complementary strand and counterstrand of agene transcript brings about a highly efficient suppression of theexpression of the gene in question. The phenotype which is brought aboutgreatly resembles one of a corresponding knockout mutant (Waterhouse P Met al. (1998) Proc Natl Acad Sci USA 95:13959-64).

The dsRNAi method has proved to be especially efficient and advantageousfor reducing expression. As described in WO 99/32619, inter alia, dsRNAiapproaches are markedly superior to traditional antisense approaches.

The invention therefore also relates to double-stranded RNA molecules(dsRNA molecules) which, when introduced into a plant (or a cell,tissue, organs, in particular leaf epidermis derived therefrom), bringabout the reduction of a BI-1.

In the double-stranded RNA molecule for reducing the expression of aBI-1 protein,

-   a) one of the two RNA strands is essentially identical with at least    a part of a BI-1 nucleic acid sequence, and-   b) the respective other RNA strand is essentially identical with at    least a part of the complementary strand of a BI-1 nucleic acid    sequence.

In a furthermore preferred embodiment, the double-stranded RNA moleculefor reducing the expression of a BI-1 protein comprises:

-   a) a sense RNA strand comprising at least one ribo-nucleotide    sequence which is essentially identical with at least a part of the    sense RNA transcript of a nucleic acid sequence coding for a BI-1    protein, and-   b) an antisense RNA strand which is essentially—preferably    fully—complementary to the RNA sense strand of a).

With regard to the double-stranded RNA molecules, BI-1 nucleic acidsequence preferably refers to a sequence of SEQ ID NO: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 38 or a functionalequivalent of the same.

“Essentially identical”-means that the dsRNA sequence can also compriseinsertions, deletions and individual point mutations in comparison withthe BI-1 target sequence while still efficiently bringing about areduction of the expression. Preferably, the homology between the sensestrand of an inhibitory dsRNA and a partial segment of a BI-1 nucleicacid sequence (or between the antisense strand and the complementarystrand of a BI-1 nucleic acid sequence) as defined above amounts to atleast 50% or 75%, preferably to at least 80%, very especially preferablyto at least 90%, most preferably to 100%.

The length of the partial segment amounts to at least 10 bases,preferably to at least 25 bases, especially preferably to at least 50bases, very especially preferably to at least 100 bases, most preferablyto at least 200 bases or at least 300 bases. As an alternative, an“essentially identical” dsRNA can also be defined as a nucleic acidsequence which is capable of hybridizing with a part of a BI-1 genetranscript (for example in 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at50° C. or 70° C. for 12 to 16 hours).

“Essentially complementary” means that the antisense RNA strand may alsocomprise insertions, deletions and individual point mutations incomparison with the complement of the sense RNA strand. The homologybetween the antisense RNA strand and the complement of the sense RNAstrand preferably amounts to at least 80%, preferably to at least 90%,very especially preferably to at least 95%, most preferably to 100%.

“Part of the sense RNA transcript” of a nucleic acid sequence coding fora BI-1 protein or a functional equivalent thereof refers to fragments ofan RNA or mRNA transcribed from a nucleic acid sequence coding for aBI-1 protein or a functional equivalent thereof, preferably from a BI-1gene. In this context, the fragments preferably have a sequence lengthof at least 2.0 bases, preferably at least 50 bases, especiallypreferably at least 100 bases, very especially preferably at least 200bases, most preferably at least 500 bases. Also comprised is thecomplete transcribed RNA or mRNA.

Also comprised is the use of the dsRNA molecules according to theinvention in the methods according to the invention for generating apathogen resistance in plants.

The dsRNA can consist of one or more strands of polymerizedribonucleotides. Furthermore, modifications of both the sugar-phosphatebackbone and of the nucleotides may be present. For example, thephosphodiester bonds of the natural RNA can be modified in such a waythat they comprise at least one nitrogen or sulfur hetero atom. Basescan be modified in such a way that the activity of, for example,adenosine deaminase is limited. These and further modifications aredescribed further below in the methods for stabilizing antisense RNA.

To achieve the same purpose, it is naturally also possible to introduce,into the cell or the organism, a plurality of individual dsRNAmolecules, each of which comprises one of the above-definedribonucleotide sequence segments.

The dsRNA can be prepared enzymatically or fully or partially bychemical synthesis.

The double-stranded dsRNA structure can be formed starting from twocomplementary, separate RNA strands or—preferably—starting from a singleautocomplementary RNA strand.

The double-stranded structure can be formed starting from a singleautocomplementary strand or starting from two complementary strands. Inthe case of a single auto-complementary strand, sense and antisensesequences can be linked by a linking sequence (“linker”) and form forexample a hairpin structure. The linking sequence can preferably be anintron, which is spliced out after the dsRNA has been synthesized. Thenucleic acid sequence coding for a dsRNA can comprise further elementssuch as, for example, transcription termination signals orpolyadenylation signals. If the two strands of the dsRNA are to becombined in a cell or plant, this can be effected in different ways:

The nucleic acid sequence coding for a dsRNA can comprise furtherelements, such as, for example, transcription termination signals orpolyadenylation signals.

If the two strands of the dsRNA are to be combined in a cell or plant,this can be effected in different ways:

-   a) transformation of the cell or plant with a vector which comprises    both expression cassettes,-   b) cotransformation of the cell or plant with two vectors, one    comprising the expression cassettes with the sense strand, the other    comprising the expression cassettes with the antisense strand.-   c) Hybridization of two plants, each of which has been transformed    with one vector, one comprising the expression cassettes with the    sense strand, the other comprising the expression cassettes with the    antisense strand.

The formation of the RNA duplex can be initiated either externally ofthe cell or within the same. As in WO 99/53050, the dsRNA can alsocomprise a hairpin structure by linking sense and antisense strands by alinker (for example an intron). The autocomplementary dsRNA structuresare preferred since they only require the expression of one constructand always comprise the complementary strands in an equimolar ratio.

The expression cassettes encoding the antisense or sense strand of adsRNA or the autocomplementary strand of the dsRNA are preferablyinserted, under the control of an epidermis-specific promoter asdetailed herein, into a vector and, using the methods describedhereinbelow, stably inserted into the genome of a plant in order toensure permanent expression of the dsRNA in the epidermis, usingselection markers for example.

The dsRNA can be introduced using a quantity which allows at least onecopy per cell. Greater quantities (for example at least 5, 10, 100, 500or 1000 copies per cell) may bring about a more effective reduction, ifappropriate.

As already described, 100% sequence identity between dsRNA and a BI-1gene transcript or the gene transcript of a functionally equivalent geneis not necessarily required in order to bring about an effectivereduction of the expression of BI-1. Accordingly, there is the advantagethat the method is tolerant with regard to sequence deviations as mayexist as the consequence of genetic mutations, polymorphisms orevolutionary divergences. Thus, for example, it is possible to use thedsRNA generated on the basis of the BI-1 sequence of one organism tosuppress the expression of BI-1 in another organism. The high sequencehomology between the BI-1 sequences from rice, maize and barley allowsthe conclusion that this protein is conserved to a high degree withinplants, so that the expression of a dsRNA derived from one of thedisclosed BI-1 sequences as shown in SEQ ID NO: 1, 3 or 5 appears tohave an advantageous effect in other plant species as well.

Furthermore, owing to the high homology between the individual BI-1proteins and their functional equivalents, it is possible using a singledsRNA generated from a certain BI-1 sequence of an organism to suppressthe expression of further homologous BI-1 proteins and/or theirfunctional equivalents of the same organism or else the expression ofBI-1 proteins in other related species. For this purpose, the dsRNApreferably comprises sequence regions of BI-1 gene transcripts whichcorrespond to conserved regions. Said conserved regions can easily befound by comparing sequences.

The dsRNA can be synthesized either in vivo or in vitro. To this end, aDNA sequence coding for a dsRNA can be brought into an expressioncassette under the control of at least one genetic control element (suchas, for example, promoter, enhancer, silencer, splice donor or spliceacceptor or polyadenylation signal), an epidermis-specific expression ofthe dsRNA being desired. Suitable advantageous constructions aredescribed hereinbelow. Polyadenylation is not required, nor do elementsfor initiating translation have to be present.

A dsRNA can be synthesized chemically or enzymatically. Cellular RNApolymerases or bacteriophage RNA polymerases (such as, for example, T3,T7 or SP6 RNA polymerase) can be used for this purpose. Suitable methodsfor expression of RNA in vitro are described (WO 97/32016; U.S. Pat. No.5,593,874; U.S. Pat. No. 5,698,425, U.S. Pat. No. 5,712,135, U.S. Pat.No. 5,789,214, U.S. Pat. No. 5,804,693). A dsRNA which has beensynthesized in vitro chemically or enzymatically can be isolatedcompletely or to some degree from the reaction mixture, for example byextraction, precipitation, electrophoresis, chromatography orcombinations of these methods, before being introduced into a cell,tissue or organism. The dsRNA can be introduced directly into the cellor else be applied extracellularly (for example into the interstitialspace).

However, it is preferred to transform the plant stably with anexpression construct which brings about the expression of the dsRNA inthe epidermis. Suitable methods are described hereinbelow.

b) Introduction of a BI-1 Antisense Nucleic Acid Molecule

Methods for suppressing a specific protein by preventing its mRNA fromaccumulating by means of antisense technology have been described inmany instances, including in the case of plants (Sheehy et al. (1988)Proc Natl Acad Sci USA 85: 8805-8809; U.S. Pat. No. 4,801,340; Mol J Net al. (1990) FEBS Lett 268(2):427-430). The antisense nucleic acidmolecule hybridizes, or binds, with the cellular mRNA and/or genomic DNAencoding the BI-1 target protein to be suppressed. This suppresses thetranscription and/or translation of the target protein. Hybridizationcan originate conventionally by the formation of a stable duplex or—inthe case of genomic DNA—by the antisense nucleic acid molecule bindingto the duplex of the genomic DNA by specific interaction in the majorgroove of the DNA helix. The introduction is effected in such a way thatthe amount or function of BI-1 is reduced specifically in the epidermis,for example by transient transformation of the epidermis or stabletransformation under the expressional control of a suitable constructwith an epidermis-specific promoter.

An antisense nucleic acid sequence suitable for reducing a BI-1 proteincan be deduced using the nucleic acid sequence encoding this protein,for example the nucleic acid sequence as shown in SEQ ID NO: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 38, or coding for afunctional equivalent thereof, following Watson and Crick's base-pairingrules. The antisense nucleic acid sequence can be complementary to allof the transcribed mRNA of said protein, be limited to the codingregion, or else only be composed of an oligonucleotide, which ispartially complementary to the coding or noncoding sequence of the mRNA.Thus, for example, the oligonucleotide can be complementary to theregion comprising the translation start for said protein. Antisensenucleic acid sequences can be, for example, 5, 10, 15, 20, 25, 30, 35,40, 45 or 50 nucleotides in length, but may also be longer and compriseat least 100, 200, 500, 1000, 2000 or 5000 nucleotides. Antisensenucleic acid sequences can be expressed recombinantly or synthesizedchemically or enzymatically using methods known to the skilled worker.In the case of chemical synthesis, natural or modified nucleotides maybe used. Modified nucleotides can impart an increased biochemicalstability to the antisense nucleic acid sequence and lead to anincreased physical stability of the duplex formed of antisense nucleicacid sequence and sense target sequence. The following can be used: forexample phosphorothioate derivatives and acridine-substitutednucleotides such as 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthin, xanthin, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxy-methylaminomethyl-2-thiouridine,5-carboxymethyl-aminomethyluracil, dihydrouracil,β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine,1-methyl-inosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine,3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine,5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,β-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid,pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethyl ester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl)uracil and 2,6-diaminopurine.

In a further preferred embodiment, the expression of a BI-1 protein canbe inhibited by nucleotide sequences which are complementary to theregulatory region of a BI-1 gene (for example a BI-1 promoter and/orenhancer) and which form triple-helical structures with that DNA doublehelix so that the transcription of the BI-1 gene is reduced. Suchmethods have been described (Helene C (1991) Anticancer Drug Res6(6):569-84; Helene C et al. (1992) Ann NY Acad Sci 660:27-36; Maher L J(1992) Bioassays 14(12):807-815).

In a further embodiment, the antisense nucleic acid molecule can be anα-anomeric nucleic acid. Such α-anomeric nucleic acid molecules formspecific double-stranded hybrids with complementary RNA in which—asopposed to the conventional β-nucleic acids—the two strands run parallelto one another (Gautier C et al. (1987) Nucleic Acids Res 15:6625-6641).The antisense nucleic acid molecule can furthermore also comprise2′-O-methylribonucleotides (Inoue et al. (1987) Nucleic Acids Res15:6131-6148) or chimeric RNA/DNA analogs (Inoue et al. (1987) FEBS Lett215:327-330).

c) Introduction of a BI-1 Antisense Nucleic Acid Molecule in Combinationwith a Ribozyme

The above-described antisense strategy can be combined advantageouslywith a ribozyme method. Catalytic RNA molecules or ribozymes can beadapted to suit any target RNA and cleave the phosphodiester backbone atspecific positions, functionally deactivating the target RNA (Tanner N K(1999) FEMS Microbiol Rev 23(3):257-275). The ribozyme itself is notmodified thereby, but is capable of cleaving further target RNAmolecules analogously, thereby assuming the qualities of an enzyme. Theincorporation of ribozyme sequences into antisense RNAs confers thisenzyme-like RNA-cleaving quality to precisely these antisense RNAs, thusincreasing their efficacy in inactivating the target RNA. The generationand the use of such ribozyme antisense RNA molecules is described, forexample, in Haselhoff et al. (1988) Nature 334: 585-591.

In this manner, ribozymes (for example “hammerhead” ribozymes; Haselhoffand Gerlach (1988) Nature 334:585-591) can be used catalytically tocleave the mRNA of an enzyme to be suppressed, for example BI-1, and toprevent translation. The ribozyme technique can increase the efficacy ofan antisense strategy. Methods of expressing ribozymes for reducingspecific proteins are described in EP 0 291 533, EP 0 321 201, EP 0 360257. The expression of ribozyme in plant cells has also been described(Steinecke P et al. (1992) EMBO J 11(4):1525-1530; de Feyter R et al.(1996) Mol Gen Genet. 250(3):329-338). Suitable target sequences andribozymes can be determined as described for example by “Steinecke P,Ribozymes, Methods in Cell Biology 50, Galbraith et al. eds, AcademicPress, Inc. (1995), pp. 449 460” by calculating the secondary structureof ribozyme RNA and target RNA as well as by their interaction (Bayley CC et al. (1992) Plant Mol. Biol. 18(2):353-361; Lloyd A M and Davis R Wet al. (1994) Mol Gen Genet. 242(6):653-657). For example, derivativesof the Tetrahymena L-19 IVS RNA with regions which are complementary tothe mRNA of the BI-1 protein to be suppressed can be constructed (seealso U.S. Pat. No. 4,987,071 and U.S. Pat. No. 5,116,742). As analternative, such ribozymes can also be identified from a library ofdiverse ribozymes via a selection process (Bartel D and Szostak J W(1993) Science 261:1411-1418). Expression takes place, for example,under the control of an epidermis-specific promoter.

d) Introduction of a BI-1 Sense Nucleic Acid Molecule for InducingCosuppression

The epidermis-specific expression of a BI-1 nucleic acid molecule insense orientation can lead to cosuppression, in the epidermis cells, ofthe corresponding homologous endogenous gene. The expression of senseRNA with homology with an endogenous gene can reduce or switch off theexpression of the former, similarly to what has been described forantisense approaches (Jorgensen et al. (1996) Plant Mol Biol31(5):957-973; Goring et al. (1991) Proc Natl Acad Sci USA 88:1770-1774;Smith et al. (1990) Mol Gen Genet 224:447-481; Napoli et al. (1990)Plant Cell 2:279-289; Van der Krol et al. (1990) Plant Cell 2:291-99).In this context, the homologous gene to be reduced can be representedeither fully or only in part by the construct introduced. Thepossibility of translation is not required. The application of thistechnique to plants is described, for example, by Napoli et al. (1990)The Plant Cell 2: 279-289 and in U.S. Pat. No. 5,034,323.

The cosuppression is preferably realized by using a sequence essentiallyidentical with at least a part of the nucleic acid sequence coding for aBI-1 protein or a functional equivalent thereof, for example the nucleicacid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33 or 38 or the nucleic acid sequence coding fora functional equivalent thereof.

e) Introduction of Nucleic Acid Molecules Coding for a Dominant-NegativeBI-1 Protein

The function or activity of a BI-1 protein can also be reducedefficiently in epidermis cells by expressing, in an epidermis-specificmanner, a dominant-negative variant of this BI-1 protein. Methods ofreducing the function or activity of a protein by coexpressing itsdominant-negative form are known to the skilled worker (Lagna G andHemmati-Brivanlou A (1998) Current Topics in Developmental Biology36:75-98; Perlmutter R M and Alberola-Ila J (1996) Current Opinion inImmunology 8(2):285-90; Sheppard D (1994) American Journal ofRespiratory Cell & Molecular Biology. 11(1):1-6; Herskowitz I (1987)Nature 329(6136):219-22).

The amino acid which is preferably to be mutated in BI-1 homologs fromother species can be determined for example by computer-aided comparison(“alignment”). These mutations for achieving a dominant-negative BI-1variant are preferably carried out at the level of the nucleic acidsequence coding for BI-1 proteins. A corresponding mutation can bebrought about for example by PCR-mediated in-vitro mutagenesis usingsuitable oligonucleotide primers, by which the desired mutation isintroduced. This is done using methods known to the skilled worker; forexample, the “LA PCR in vitro Mutagenesis Kit” (Takara Shuzo, Kyoto) maybe used for this purpose. A method of generating a dominant-negativevariant of a maize RacB protein is also described in WO 00/15815(Example 4, p. 69).

Such a mutant can then be expressed for example under the control of anepidermis-specific promoter.

f) Introduction of DNA- or Protein-Binding Factors Against BI-1 Genes,BI-1 RNAs or BI-1 Proteins

BI-1 gene expression in the epidermis may also be reduced using specificDNA-binding factors, for example factors of the zinc fingertranscription factor type. These factors attach to the genomic sequenceof the endogenous target gene, preferably in the regulatory regions, andbring about repression of the endogenous gene. The use of such a methodmakes the reduction of the expression of an endogenous BI-1 genepossible without it being necessary to recombinantly manipulate itssequence. Suitable methods for the preparation of suitable factors havebeen described (Dreier B et al. (2001) J Biol Chem 276(31):29466-78;Dreier B et al. (2000) J Mol Biol 303(4):489-502; Beerli R R et al.(2000) Proc Natl Acad Sci USA 97 (4):1495-1500; Beerli R R et al. (2000)J Biol Chem 275(42):32617-32627; Segal D J and Barbas C F 3rd. (2000)Curr Opin Chem Biol 4(1):34-39; Kang J S and Kim J S (2000) J Biol Chem275(12):8742-8748; Beerli R R et al. (1998) Proc Natl Acad Sci USA95(25):14628-14633; Kim J S et al. (1997) Proc Natl Acad Sci USA94(8):3616-3620; Klug A (1999) J Mol Biol 293(2):215-218; Tsai S Y etal. (1998) Adv Drug Deliv Rev 30(1-3):23-31; Mapp A K et al. (2000) ProcNatl Acad Sci USA 97(8):3930-3935; Sharrocks A D et al. (1997) Int JBiochem Cell Biol 29(12):1371-1387; Zhang L et al. (2000) J Biol Chem275(43):33850-33860).

These factors can be selected using any desired portion of a BI-1 gene.This segment is preferably located in the promoter region. For genesuppression, however, it may also be in the region of the coding exonsor introns. The segments in question can be obtained by the skilledworker from Genbank by database search or, starting from a BI-1 cDNAwhose gene is not present in Genbank, by screening a genomic library forcorresponding genomic clones. The skilled worker is familiar with themethods required therefor, for example, these factors can be expressedunder the control of an epidermis-specific promoter or other factorswhich mediate epidermis-specific expression.

Furthermore, it is possible to introduce, into a cell, factors whichinhibit the BI-1 target protein itself. The protein-binding factors canbe, for example, aptamers (Famulok M and Mayer G (1999) Curr TopMicrobiol Immunol 243:123-36) or antibodies or antibody fragments orsingle-chain antibodies. Methods for obtaining these factors have beendescribed and are known to the skilled worker. For example, acytoplasmic scFv antibody was employed to modulate the activity of thephytochrome A protein in genetically modified tobacco plants (Owen M etal. (1992) Biotechnology (N Y) 10(7):790-794; Franken E et al. (1997)Curr Opin Biotechnol 8(4):411-416; Whitelam (1996) Trend Plant Sci1:286-272).

Gene expression may also be suppressed by tailor-madelow-molecular-weight synthetic compounds, for example of the polyamidetype (Dervan P B and Bürli R W (1999) Current Opinion in ChemicalBiology 3:688-693; Gottesfeld J M et al. (2000) Gene Expr 9(1-2):77-91).These oligomers are composed of the units 3-(dimethylamino)propylamine,N-methyl-3-hydroxypyrrole, N-methylimidazole and N-methylpyrrole and canbe adapted to any piece of double-stranded DNA in such a way that theybind into the major groove in a sequence-specific manner and block theexpression of these gene sequences. Suitable methods have been described(see, inter alia, Bremer R E et al. (2001) Bioorg Med. Chem.9(8):2093-103; Ansari A Z et al. (2001) Chem Biol. 8(6):583-92;Gottesfeld J M et al. (2001) J Mol. Biol. 309(3):615-29; Wurtz N R etal. (2001) Org Lett 3(8):1201-3; Wang C C et al. (2001) Bioorg Med Chem9(3):653-7; Urbach A R and Dervan P B (2001) Proc Natl Acad Sci USA98(8):4343-8; Chiang S Y et al. (2000) J Biol. Chem. 275(32):24246-54).

All the abovementioned factors are introduced in an epidermis-specificmanner in order to ensure a reduction of the BI-1 activity only inepidermal cells, for example by means of expression under the control ofan epidermis-specific promoter as they are mentioned for examplehereinabove.

g) Introduction of Viral Nucleic Acid Molecules and CorrespondingExpression Constructs which Cause the Degradation of BI-1 RNA

BI-1 expression in the epidermis can also be brought about efficientlyby inducing the specific degradation of BI-1 RNA in epidermal cells withthe aid of a viral expression system (amplicon) (Angell, S M et al.(1999) Plant J. 20(3):357-362). These systems—also termed “VIGS” (viralinduced gene silencing)—introduce, into the plant, nucleic acidmolecules with homology to the transcripts to be suppressed, with theaid of viral vectors. Then, transcription is switched off, probablymediated by plant defense mechanisms against viruses. Suitabletechniques and methods have been described (Ratcliff F et al. (2001)Plant J 25(2):237-45; Fagard M and Vaucheret H (2000) Plant Mol Biol43(2-3):285-93; Anandalakshmi R et al. (1998) Proc Natl Acad Sci USA95(22):13079-84; Ruiz M T (1998) Plant Cell 10(6): 937-46).

h) Introduction of Constructs for Inducing Homologous Recombination atEndogenous BI-1 Genes, for Example for Generating Knockout Mutants

An example of what is used for generating a homologously recombinantorganism with reduced BI-1 activity in the epidermal cells is a nucleicacid construct comprising at least a part of an endogenous BI-1 genewhich is modified by a deletion, addition or substitution of at leastone nucleotide in such a way that its functionality is reduced or fullydestroyed. The modification may also relate to the regulatory elements(for example the promoter) of the gene, so that the coding sequenceremains unmodified, but expression (transcription and/or translation)does not take place and is reduced.

In the case of conventional homologous recombination, the modifiedregion is flanked at its 5′ and 3′ ends by further nucleic acidsequences which must be sufficient in length for making recombinationpossible. They are, as a rule, in the range of several hundred bases toseveral kilobases in length (Thomas K R and Capecchi M R (1987) Cell51:503; Strepp et al. (1998) Proc Natl Acad Sci USA 95(8): 4368-4373).For homologous recombination, the host organism—for example a plant—istransformed with the recombination construct using the methods describedhereinbelow, and clones which have successfully undergone recombinationare selected, for example using a resistance to antibiotics orherbicides.

Homologous recombination is a relatively rare event in highereukaryotes, especially in plants. Random integrations into the hostgenome predominate. One possibility of eliminating the randomlyintegrated sequences and thus increasing the number of cell clones witha correct homologous recombination is the use of a sequence-specificrecombination system as described in U.S. Pat. No. 6,110,736, by whichunspecifically integrated sequences can be deleted again, whichsimplifies the selection of events which have integrated successfullyvia homologous recombination. A large number of sequence-specificrecombination systems can be used, examples being the Cre/lox system ofbacteriophage P1, the FLP/FRT system of yeast, the Gin recombinase ofphage Mu, the Pin recombinase from E. coli, and the R/RS system of thepSR1 plasmid. The bacteriophage P1 Cre/10× and the yeast FLP/FRT systemare preferred. The FLP/FRT and cre/lox recombinase system has alreadybeen applied to plant systems (Odell et al. (1990) Mol Gen Genet 223:369-378). Epidermis-specific recombination can be ensured for example bythe expression of the systems and enzymes which mediate recombinationtaking place under the control of an epidermis-specific promoter.

i) Introduction of Mutations into Endogenous BI-1 Genes for Generating aLoss of Function (for Example Generation of Stop Codons, Reading-FrameShifts and the Like)

Further suitable methods for reducing the BI-1 activity are theintroduction of nonsense mutations into endogenous BI-1 genes, forexample by introducing RNA/DNA oligonucleotides into the epidermal cells(Zhu et al. (2000) Nat Biotechnol 18(5):555-558) and the generation ofknockout mutants with the aid of, for example, T-DNA mutagenesis (Konczet al. (1992) Plant Mol Biol 20(5):963-976), ENU (N-ethyl-N-nitrosourea)mutagenesis or homologous recombination (Hohn B and Puchta (1999) H ProcNatl Acad Sci USA 96:8321-8323). Point mutations can also be generatedby means of DNA-RNA hybrids also known under the name “chimeraplasty”(Cole-Strauss et al. (1999) Nucl Acids Res 27(5):1323-1330; Kmiec (1999)Gene therapy American Scientist 87(3):240-247).

The methods of dsRNAi, cosuppression by means of sense RNA and “VIGS”(“virus induced gene silencing”) are also termed “post-transcriptionalgene silencing” (PTGS). PTGS methods, like the reduction of the BI-1function or activity with dominant-negative BI-1 variants, areespecially advantageous because the demands regarding the homologybetween the endogenous gene to be suppressed and the sense or dsRNAnucleic acid sequence expressed recombinantly (or between the endogenousgene and its dominant-negative variant) are lower than, for example, inthe case of a traditional antisense approach. Such criteria with regardto homology are mentioned in the description of the dsRNAi method andcan generally be applied to PTGS methods or dominant-negativeapproaches. Thus, using the BI-1 nucleic acid sequences, it ispresumably also possible efficiently to suppress the expression ofhomologous BI-1 proteins in other species without the isolation andstructure elucidation of the BI-1 homologs occurring therein beingrequired. Considerably less labor is therefore required. Similarly, theuse of dominant-negative variants of a BI-1 protein can presumably alsoefficiently reduce or suppress the function/activity of its homolog inother plant species.

All of the substances and compounds which directly or indirectly bringabout a reduction in protein quantity, RNA quantity, gene activity orprotein activity of a BI-1 protein shall subsequently be combined in theterm “anti-BI-1” compounds. The term “anti-BI-1” compound explicitlyincludes the nucleic acid sequences, peptides, proteins or other factorsemployed in the above-described methods.

For the purposes of the invention, “introduction” comprises all of themethods which are capable of directly or indirectly introducing an“anti-BI-1” compound into the epidermis or a substantial number of theepidermal cells, compartments or tissues thereof, or of generating sucha compound there. Direct and indirect methods are comprised. Theintroduction can lead to a transient presence of an “anti-BI-1” compound(for example of a dsRNA) or else to its stable presence.

Owing to the different nature of the above-described approaches, the“anti-BI-1” compound can exert its function directly (for example byinsertion into an endogenous BI-1 gene). However, its function can alsobe exerted indirectly following transcription into an RNA (for examplein the case of antisense approaches) or following transcription andtranslation into a protein (for example in the case of binding factors).The invention comprises both directly and indirectly acting “anti-BI-1”compounds.

The term “introducing” comprises for example methods such astransfection, transduction or transformation.

The term “anti-BI-1” compounds therefore also comprises recombinantexpression constructs, for example, which bring about an expression(i.e. transcription and, if appropriate, translation), for example of aBI-1 dsRNA or a BI-1 “antisense” RNA in an epidermis-specific manner.

In said expression constructs, a nucleic acid molecule whose expression(transcription and, if appropriate, translation) generates an“anti-BI-1” compound is preferably operably linked to at least onegenetic control element (for example a promoter) which ensuresexpression in an organism, preferably in plants, and preferably in anepidermis-specific manner. If the expression construct is to beintroduced directly into the plant and the “anti-BI-1” compound (forexample the BI-1 dsRNA) is to be generated therein in planta,plant-specific genetic control elements (for example promoters) arepreferred, where, as the result of what has been said above, theepidermis-specific activity of the promoter is mandatory in mostembodiments for an epidermis-specific reduction of BI-1, as describedabove. However, the “anti-BI-1” compound may also be generated in otherorganisms or in vitro and then be introduced into the plant. Preferredin this context are all of the prokaryotic or eukaryotic genetic controlelements (for example promoters) which permit the expression in theorganism chosen in each case for the preparation.

Functional linkage is to be understood as meaning, for example, thesequential arrangement of a promoter with the nucleic acid sequence tobe expressed (for example an “anti-BI-1” compound) and, if appropriate,further regulatory elements such as, for example, a terminator in such away that each of the regulatory elements can fulfill its function whenthe nucleic acid sequence is expressed recombinantly, depending on thearrangement of the nucleic acid sequences in relation to sense orantisense RNA. To this end, direct linkage in the chemical sense is notnecessarily required. Genetic control sequences such as, for example,enhancer sequences, can also exert their function on the target sequencefrom positions which are further away, or indeed from other DNAmolecules (localization in cis and trans respectively). Preferredarrangements are those in which the nucleic acid sequence to beexpressed recombinantly is positioned behind the sequence acting aspromoter, so that the two sequences are linked covalently to each other.The distance between the promoter sequence and the nucleic acid sequenceto be expressed recombinantly is preferably less than 200 base pairs,especially preferably less than 100 base pairs, very especiallypreferably less than 50 base pairs.

Functional linkage, and an expression cassette, can be generated bymeans of customary recombination and cloning techniques as aredescribed, for example, in Maniatis T, Fritsch E F and Sambrook J (1989)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor (NY), in Silhavy T J, Berman M L and Enquist L W(1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory,Cold Spring Harbor (NY), in Ausubel F M et al. (1987) Current Protocolsin Molecular Biology, Greene Publishing Assoc. and Wiley Interscienceand in Gelvin et al. (1990) In: Plant Molecular Biology Manual. However,further sequences which, for example, act as a linker with specificcleavage sites for restriction enzymes, or as a signal peptide, may alsobe positioned between the two sequences. The insertion of sequences mayalso lead to the expression of fusion proteins. Preferably, theexpression cassette, consisting of a linkage of promoter and nucleicacid sequence to be expressed, can exist in a vector-integrated form andbe inserted into a plant genome, for example by transformation. Thecontrol elements preferably mediate an epidermis-specific expression.

The abovementioned methods (a) to (i) can also be employed for thereduction of the activity or function, in particular the expression, ofthe other proteins mentioned herein, in particular for the reduction ofMLO, RacB and NaOx.

The barley BI1 protein (hvBI1) is predominantly expressed in themesophyll (Example 6) and is upregulated as the result of infection withBlumeria (syn. Erysiphe) graminis f. sp. hordei (Example 2). Therecombinant mesophyll-specific overexpression in mlo-resistant barleyleads not only to resistance to in particular necrotrophic andhemibiotrophic pathogens, but also to a plant which is resistant toBlumeria (syn. Erysiphe) graminis f. sp. hordei and which exhibits nonecrotic lesions (“mlo lesions”; negative side effect of the mloresistance). Utilizing this effect, the negative side effects of themlo-mediated resistance (yield loss of approximately 5%, Jorgensen J H(1992) Euphytica 63: 141-152; hypersusceptibility to necrotrophic fungi,Jarosch B et al. (1999) Mol Plant Microbe Interact 12:508-514; Kumar. Jet al. (2001) Phytopathology 91:127-133) can be suppressed withoutadversely affecting the mlo resistance itself.

Furthermore, it can be demonstrated in a surprising manner that anoverexpression of BI1 results in resistance to stress factors likeagents which trigger necroses (isolated for example from necrotrophicharmful fungi; Example 2).

Accordingly, the method according to the invention offers an efficientbiotechnological strategy for resistance to the formation of necroses asthe result of endogenous, abiotic and biotic stress, for example mlolesions, ozone damage, necrotrophic and hemibiotrophic harmfulorganisms.

BI1 proteins appear to be crucial regulators of the race-unspecificfungal resistance in plants. This makes possible their broad use inbiotechnological strategies for increasing the pathogen resistance inplants, in particular fungal resistance. The method according to theinvention can be applied to all plant species, in principle. BI1proteins have been identified in a large number of plants, both monocotsand dicots (see above).

For the purposes of the present invention, “approximately”, whenreferring to numbers or sizes, means a range of numbers or sizes aroundthe stated value of the number or size. In general, the termapproximately means a range of in each case 20% above and below of thevalue stated.

The term “plant” as used herein refers to all genera and species ofhigher and lower plants of the Plant Kingdom. The term includes themature plants, seed, shoots and seedlings and their derived parts,propagation material, plant organs, tissue, protoplasts, callus andother cultures, for example cell cultures, and any other types ofplant-cell associations to give functional or structural units. The termmature plants refers to plants at any desired developmental stage beyondthat of the seedling. Seedling refers to a young immature plant at anearly developmental stage.

“Plant” comprises all annual and perennial monocotyledonous anddicotyledonous plants and includes by way of example but not bylimitation those of the genera Cucurbita, Rosa, Vitis, Juglans,Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna,Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica,Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon,Nicotiana, Solarium, Petunia, Digitalis, Majorana, Cichorium,Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis,Nemesis, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio,Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium,Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum, Picea andPopulus.

The term “plant” preferably comprises monocotyledonous crop plants suchas, for example, cereal species such as wheat, barley, millet, rye,triticale, maize, rice, sorghum or oats and also sugar cane.

The term furthermore comprises dicotyledonous crop plants such as, forexample,

-   -   Brassicaceae such as oilseed rape, turnip rape, cress,        Arabidopsis, cabbage species,    -   Leguminosae such as soybean, alfalfa, pea, beans or peanut,    -   Solanaceae such as potato, tobacco, tomato, egg plant or        capsicum,    -   Asteraceae such as sunflower, Tagetes, lettuce or Calendula,    -   Cucurbitaceae such as melon, pumpkin/squash or zucchini,        and also linseed (flax), cotton, hemp, clover, spinach, red        pepper, carrot, beet, radish, sugar beet, sweet potato,        cucumber, chicory, cauliflower, broccoli, asparagus, onion,        garlic, celery/celeriac, strawberry, raspberry, blackberry,        pineapple, avocado and the various tree, bush, nut and vine        species. Tree species preferably comprise plum, cherry, peach,        nectarine, apricot, banana, papaya, mango, apple, pear, quince.

Preferred within the scope of the invention are those plants which areemployed as foodstuffs or feeding stuffs, very especially preferablymonocotyledonous genera and agriculturally important species such aswheat, oats, millet, barley, rye, maize, rice, buckwheat, sorghum,triticale, spelt, linseed or sugar cane.

For the purposes of the present invention, the term “stress factor”comprises biotic stress factors (such as in particular the pathogensdetailed hereinbelow) and abiotic stress factors. Abiotic stress factorswhich may be mentioned by way of example, but not by limitation, are:chemical stress (for example caused by agrochemicals and/orenvironmental chemicals), UV radiation, heat, cold, drought, increasedhumidity.

“Stress resistance” means the reduction or alleviation of symptoms of aplant as a result of stress. The symptoms can be manifold, butpreferably comprise those which directly or indirectly have an adverseeffect on the quality of the plant, the quantity of its yield, itssuitability for use as feeding stuff or foodstuff, or else make sowing,planting, harvesting or processing of the crop more difficult.

“Pathogen resistance” denotes the reduction or alleviation of diseasesymptoms of a plant following infection by at least one pathogen. Thesymptoms can be manifold, but preferably comprise those which directlyor indirectly have an adverse effect on the quality of the plant, thequantity of the yield, its suitability for use as feeding stuff orfoodstuff, or else which make sowing, planting, harvesting or processingof the crop more difficult.

“Conferring”, “generating” or “increasing” a pathogen resistance and“the existence of” a pathogen resistance means that the defensemechanisms of a specific plant species or variety are increasinglyresistant to one or more stress factors or pathogens due to the use ofthe method according to the invention in comparison with the wild typeof the plant (“starting plant”), to which the method according to theinvention has not been applied, under otherwise identical conditions(such as, for example, climatic conditions, growing conditions, type ofstress or pathogen species and the like). The increased resistancemanifests itself preferably in a reduced manifestation of the stress ordisease symptoms, disease symptoms comprising—in addition to theabovementioned adverse effects—for example also the penetrationefficiency of a pathogen into the plant or plant cells or theproliferation efficiency in or on the same. In this context, the stressor disease symptoms are preferably reduced by at least 10% or at least20%, especially preferably by at least 40% or 60%, very especiallypreferably by at least 70% or 80% and most preferably by at least 90% or95%.

“Selection” with regard to plants in which—as opposed or as compared tothe starting plant—resistance to at least one stress factor or pathogenexists or is increased means all those methods which a are suitable forrecognizing an existing or increased resistance to stress or pathogens.For example, these may be symptoms of pathogen infection (for examplethe development of necroses in the case of fungal infection), but mayalso comprise the above-described symptoms which relate to the qualityof the plant, the quantity of the yield, the suitability for use asfeeding stuff or foodstuff and the like.

“Pathogen” within the scope of the invention means by way of example butnot by limitation viruses or viroids, bacteria, fungi, animal pests suchas, for example, insects or nematodes. Especially preferred are fungi,especially necrotrophic or hemibiotrophic fungi. However, it can beassumed that the mesophyll-specific expression of a BI1 protein alsobrings about resistance to other pathogens since an overall resistanceto stress factors is generated.

The following pathogens may be mentioned by way of example but not bylimitation:

1. Fungal Pathogens or Fungus-Like Pathogens:

Fungal pathogens or fungus-like pathogens (e.g. Chromista) arepreferably from the group comprising Plasmodiophoramycota, Oomycota,Ascomycota, Chytridiomycetes, Zygomycetes, Basidiomycota andDeuteromycetes (Fungi imperfecti). The pathogens mentioned in Tables 1to 4 and the diseases with which they are associated may be mentioned byway of example but not by limitation. The following English and Germanterms can be used interchangeably:

-   Ährenfäule—ear rot/head blight-   Stengelfäule—stalk rot-   Wurzelfäule—root rot-   Rost—rust-   Falscher Mehltau—downy mildew

Further translations can be found for example athttp://www.bba.de/english/database/psmengl/pilz.htm. TABLE 1 Diseasescaused by biotrophic phytopathogenic fungi Disease Pathogen Brown rustPuccinia recondita Yellow rust P. striiformis Powdery mildew Erysiphegraminis/Blumeria graminis Rust (common maize) Puccinia sorghi Rust(southern corn) Puccinia polysora Tobacco frogeye disease Cercosporanicotianae Rust (tropical maize) Physopella pallescens, P. zeae =Angiopsora zeae

TABLE 2 Diseases caused by necrotrophic and/or hemibiotrophic fungi andOomycetes Disease Pathogen Glume blotch Septoria (Stagonospora) nodorumLeaf blotch Septoria tritici Ear fusarioses Fusarium spp. EyespotPseudocercosporella herpotrichoides Smut Ustilago spp. Potato blightPhytophthora infestans Bunt Tilletia caries Blackleg Gaeumannomycesgraminis Anthrocnose leaf blight Colletotrichum graminicola Anthracnosestalk rot (teleomorph: Glomerella graminicola Politis); Glomerellatucumanensis (anamorph: Glomerella falcatum Went) Aspergillus ear andAspergillus flavus kernel rot Banded leaf and sheath spot Rhizoctoniasolani Kuhn = Rhizoctonia microsclerotia J. Matz (telomorph:Thanatephorus cucumeris) Black bundle disease Acremonium strictum W.Gams = Cephalosporium acremonium Auct. non Corda Black kernel rotLasiodiplodia theobromae = Botryodiplodia theobromae Borde blancoMarasmiellus sp. Brown spot (black spot, Physoderma maydis stalk rot)Cephalosporium kernel rot Acremonium strictum = Cephalosporiumacremonium Charcoal rot Macrophomina phaseolina Corticium ear rotThanatephorus cucumeris = Corticium sasakii Curvularia leaf spotCurvularia clavata, C. eragrostidis, = C. maculans (teleomorph:Cochliobolus eragrostidis), Curvularia inaequalis, C. intermedia(teleomorph: Cochliobolus intermedius), Curvularia lunata (teleomorph:Cochliobolus lunatus), Curvularia pallescens (teleomorph: Cochlioboluspallescens), Curvularia senegalensis, C. tuberculata (teleomorph:Cochliobolus tuberculatus) Didymella leaf spot Didymella exitalisDiplodia ear and stalk rot Diplodia frumenti (teleomorph: Botryosphaeriafestucae) Diplodia ear and stalk rot, Diplodia maydis = Stenocarpellaseed rot and seedling blight maydis Diplodia leaf spot or streakStenocarpella macrospora = Diplodialeaf macrospora Brown stripe downySclerophthora rayssiae var. zeae mildew Crazy top downy mildewSclerophthora macrospora = Sclerospora macrospora Green ear downy mildewSclerospora graminicola (graminicola downy mildew) Dry ear rot (cob,Nigrospora oryzae kernel and stalk rot) (teleomorph: Khuskia oryzae) Earrots/head blights Alternaria alternata = A. tenuis, (minor) Aspergillusglaucus, A. niger, Aspergillus spp., Botrytis cinerea (teleomorph:Botryotinia fuckeliana), Cunninghamella sp., Curvularia pallescens,Doratomyces stemonitis = Cephalotrichum stemonitis, Fusarium culmorum,Gonatobotrys simplex, Pithomyces maydicus, Rhizopus microsporusTiegh.,R. stolonifer = R. nigricans, Scopulariopsis brumptii Ergot(horse'stooth) Claviceps gigantea (anamorph: Sphacelia sp.) EyespotAureobasidium zeae = Kabatiella zeae Fusarium ear and stalk rot Fusariumsubglutinans = F. moniliforme var. subglutinans Fusarium kernel, rootand Fusarium moniliforme stalk rot, seed rot and (teleomorph: Gibberellafujikuroi) seedling blight Fusarium stalk rot, Fusarium avenaceumseedling root rot (teleomorph: Gibberella avenacea) Gibberella ear andstalk rot Gibberella zeae (anamorph: Fusarium graminearum) Gray earrot/head blight Botryosphaeria zeae = Physalospora zeae (anamorph:Macrophoma zeae) Gray leaf spot Cercospora sorghi = C. sorghi var.(Cercospora leaf spot) maydis, C. zeae-maydis Helminthosporium root rotExserohilum pedicellatum = Helminthosporium pedicellatum (teleomorph:Setosphaeria pedicellata) Hormodendrum ear rot/head Cladosporiumcladosporioides = Hormodendrum blight (Cladosporium rot)cladosporioides, C. herbarum (teleomorph: Mycosphaerella tassiana) Leafspots, minor Alternaria alternata, Ascochyta maydis, A. tritici, A.zeicola, Bipolaris victoriae = Helminthosporium victoriae (teleomorph:Cochliobolus victoriae), C. sativus (anamorph: Bipolaris sorokiniana =H. sorokinianum = H. sativum), Epicoccum nigrum, Exserohilum prolatum =Drechslera prolata (teleomorph: Setosphaeria prolata) Graphiumpenicillioides, Leptosphaeria maydis, Leptothyrium zeae, Ophiosphaerellaherpotricha, (anamorph: Scolecosporiella sp.), Paraphaeosphaeriamichotii, Phoma sp., Septoria zeae, S. zeicola, S. zeina Northern cornleaf blight Setosphaeria turcica (anamorph: (white blast, crown stalkExserohilum turcicum = Helminthosporium rot, stripe) turcicum) Northerncorn leaf spot Cochliobolus carbonum (anamorph: Helminthosporium ear rotBipolaris zeicola = Helminthosporium (race 1) carbonum) Blue eye, bluemold Penicillium spp., P. chrysogenum, P. expansum, P. oxalicumPhaeocytostroma stalk and Phaeocytostroma ambiguum, = Phaeocytosporellaroot rot zeae Phaeosphaeria leaf spot Phaeosphaeria maydis = Sphaerulinamaydis Physalospora ear rot/head Botryosphaeria festucae = Physalosporablight (Botryosphaeria ear zeicola (anamorph: rot/head blight) Diplodiafrumenti) Purple leaf sheath Hemiparasitic bacteria and fungiPyrenochaeta stalk and root Phoma terrestris = Pyrenochaeta rotterrestris Pythium root rot Pythium spp., P. arrhenomanes, P.graminicola Pythium stalk rot Pythium aphanidermatum = P. butleri L. Redkernel disease (ear Epicoccum nigrum mold, leaf and seed rot) Sclerotialrot Rhizoctonia zeae (teleomorph: Waitea circinata) Rhizoctonia root andstalk Rhizoctonia solani, Rhizoctonia rot zeae Root rots (minor)Alternaria alternata, Cercospora sorghi, Dictochaeta fertilis, Fusariumacuminatum (teleomorph: Gibberella acuminata), F. equiseti (teleomorph:G. intricans), F. oxysporum, F. pallidoroseum, F. poae, F. roseum, G.cyanogena, (anamorph: F. sulphureum), Microdochium bolleyi, Mucor sp.,Periconia circinata, Phytophthora cactorum, P. drechsleri, P. nicotianaevar. parasitica, Rhizopus arrhizus Rostratum leaf spot Setosphaeriarostrata, (anamorph: (Helminthosporium leaf Exserohilum rostratum =Helminthosporium disease, ear and stalk rot) rostratum) Java downymildew Peronosclerospora maydis = Sclerospora maydis Philippine downymildew Peronosclerospora philippinensis = Sclerospora philippinensisSorghum downy mildew Peronosclerospora sorghi = Sclerospora sorghiSpontaneum downy mildew Peronosclerospora spontanea = Sclerosporaspontanea Sugar cane downy mildew Peronosclerospora sacchari =Sclerospora sacchari Southern blight Sclerotium rolfsii Sacc.(teleomorph: Athelia rolfsii) Seed rot-seedling blight Bipolarissorokiniana, B. zeicola = Helminthosporium carbonum, Diplodia maydis,Exserohilum pedicillatum, Exserohilum turcicum = Helminthosporiumturcicum, Fusarium avenaceum, F. culmorum, F. moniliforme, Gibberellazeae (anamorph: F. graminearum), Macrophomina phaseolina, Penicilliumspp., Phomopsis sp., Pythium spp., Rhizoctonia solani, R. zeae,Sclerotium rolfsii, Spicaria sp. Selenophoma leaf spot Selenophoma sp.Sheath rot Gaeumannomyces graminis Shuck rot Myrothecium gramineumSilage mold Monascus purpureus, M ruber Common smut Ustilago zeae = U.maydis False smut Ustilaginoidea virens Head smut Sphacelotheca reiliana= Sporisorium holcisorghi Southern corn leaf blight Cochliobolusheterostrophus and stalk rot (anamorph: Bipolaris maydis =Helminthosporium maydis) Southern leaf spot Stenocarpella macrospora =Diplodia macrospora Stalk rots (minor) Cercospora sorghi, Fusariumepisphaeria, F. merismoides, F. oxysporum Schlechtend, F. poae, F.roseum, F. solani (teleomorph: Nectria haematococca), F. tricinctum,Mariannaea elegans, Mucor sp., Rhopographus zeae, Spicaria sp. Storagerots Aspergillus spp., Penicillium spp. and other fungi Tar spotPhyllachora maydis Trichoderma ear rot and root Trichoderma viride = T.lignorum rot teleomorph: Hypocrea sp. White ear rot, root andStenocarpella maydis = Diplodia stalk rot zeae Yellow leaf blightAscochyta ischaemi, Phyllosticta maydis (teleomorph: Mycosphaerellazeae-maydis) Zonate leaf spot Gloeocercospora sorghi

TABLE 4 Diseases caused by fungi and Oomycetes whose classification withregard to biotrophic, hemibiotrophic or necrotrophic behavior is unclearDisease Pathogen Hyalothyridium leaf spot Hyalothyridium maydis Latewilt Cephalosporium maydis

The following are especially preferred:

-   -   Plasmodiophoromycota such as Plasmodiophora brassicae        (clubroot), Spongospora subterranea, Polymyxa graminis,    -   Oomycota such as Bremia lactucae (downy mildew on lettuce),        Peronospora (downy mildew) in the case of antirrhinum (P.        antirrhini), onion (P. destructor), spinach (P. effusa), soybean        (P. manchurica), tobacco (blue mold; P. tabacina), alfalfa and        clover (P. trifolium), Pseudoperonospora humuli (downy mildew on        hops), Plasmopara (downy mildew in the case of grapes) (P.        viticola) and sunflower (P. halstedii), Sclerophtohra macrospora        (downy mildew in the case of cereals and grasses), Pythium (e.g.        blackleg on Beta beet by P. debaryanum), Phytophthora infestans        (potato blight, late blight of tomato etc.), Albugo spec.

Ascomycota such as Microdochium nivale (snow mold of rye and wheat),Fusarium graminearum, Fusarium culmorum (culm rot, inter alia of wheat),Fusarium oxysporum (Fusarium wilt of tomato), Blumeria graminis (powderymildew of barley (f.sp. hordei) and wheat (f.sp. tritici)), Erysiphepisi (powdery mildew of pea), Nectria galligena (nectria canker of fruittrees), Unicnula necator (powdery mildew of grapevine), Pseudopezizatracheiphila (red fire disease of grapevine), Claviceps purpurea (ergoton, for example, rye and grasses), Gaeumannomyces greminis (take-all onwheat, rye and other grasses), Magnaporthe grisea, Pyrenophora graminea(leaf stripe of barley), Pyrenophora teres (net blotch of barley),Pyrenophora tritici-repentis (leaf blight of wheat), Venturia inaequalis(apple scab), Sclerotinia sclerotiorum (stalk break, stem rot),Pseudopeziza medicaginis (leaf spot of alfalfa, white and red clover).

-   -   Basidiomycetes such as Typhula incarnata (typhula blight on        barley, rye, wheat), Ustilago maydis (blister smut on maize),        Ustilago nuda (loose smut on barley), Ustilago tritici (loose        smut on wheat, spelt), Ustilago avenae (loose smut on oats),        Rhizoctonia solani (rhizoctonia root rot of potato),        Sphacelotheca spp. (head smut of sorghum), Melampsora lini (rust        of flax), Puccinia graminis (stem rust of wheat, barley, rye,        oats), Puccinia recondita (leaf rust of wheat), Puccinia        dispersa (brown rust of rye), Puccinia hordei (leaf rust of        barley), Puccinia coronata (crown rust of oats), Puccinia        striiformis (yellow rust of wheat, barley, rye and a large        number of grasses), Uromyces appendiculatus (brown rust of        bean), Sclerotium rolfsii (root and stalk rots of many plants).

Deuteromycetes (Fungi imperfecti) such as Septoria (Stagonospora)nodorum (glume blotch) of wheat (Septoria tritici), Pseudocercosporellaherpotrichoides (eyespot of wheat, barley, rye), Rynchosporium secalis(leaf spot on rye and barley), Alternaria solani (early blight ofpotato, tomato), Phoma betae (blackleg on Beta beet), Cercosporabeticola (leaf spot on Beta beet), Alternaria brassicae (black spot onoilseed rape, cabbage and other crucifers), Verticillium dahliae(verticillium wilt), Colletotrichum lindemuthianum (bean anthracnose),Phoma lingam (blackleg of cabbage and oilseed rape), Botrytis cinerea(gray mold of grapevine, strawberry, tomato, hops and the like).

Most preferred are Phytophthora infestans (potato blight, brown rot intomato and the like), Microdochium nivale (previously Fusarium nivale;snow mold of rye and wheat), Fusarium graminearum, Fusarium culmorum,Fusarium avenaceum and Fusarium poae (ear rot/head blight of wheat),Fusarium oxysporum (Fusarium wilt of tomato), Magnaporthe grisea (riceblast disease), Sclerotinia sclerotium (stalk break, stem rot), Septoria(Stagonospora) nodorum and Septoria tritici (glume blotch of wheat),Alternaria brassicae (black spot on oilseed rape, cabbage and othercrucifers), Phoma lingam (blackleg of cabbage and oilseed rape).

2. Bacterial Pathogens:

The pathogens and the diseases associated with them which are mentionedin Table 5 may be mentioned by way of example but not by limitation.TABLE 5 Bacterial diseases Disease Pathogen Bacterial leaf blight andPseudomonas avenae subsp. avenae stalk rot Bacterial leaf spotXanthomonas campestris pv. holcicola Bacterial stalk rot Enterobacterdissolvens = Erwinia dissolvens Bacterial stalk and top rot Erwiniacarotovora subsp. carotovora, Erwinia chrysanthemi pv. zeae Bacterialstripe Pseudomonas andropogonis Chocolate spot Pseudomonas syringae pv.coronafaciens Goss's bacterial wilt and Clavibacter michiganensis subsp.blight (leaf freckles and nebraskensis = Corynebacterium wilt)michiganense pv. andnebraskense Holcus spot Pseudomonas syringae pv.syringae Purple leaf sheath Hemiparasitic bacteria Seed rot-seedlingblight Bacillus subtilis Stewart's disease Pantoea stewartii = Erwinia(bacterial wilt) stewartii Corn stunt Spiroplasma kunkelii(achapparramiento, maize stunt, Mesa Central or Rio Grande maize stunt)

The following pathogenic bacteria are very especially preferred:Corynebacterium sepedonicum (bacterial ring rot of potato), Erwiniacarotovora (blackleg of potato), Erwinia amylovora (fire blight of pear,apple, quince), Streptomyces scabies (potato scab), Pseudomonas syringaepv. tabaci (wildfire of tobacco), Pseudomonas syringae pv. phaseolicola(grease spot of dwarf bean), Pseudomonas syringae pv. tomato (bacterialspeck of tomato), Xanthomonas campestris pv. malvacearum (bacterialblight of cotton) and Xanthomonas campestris pv. oryzae (bacterial leafblight of rice and other grasses).

3. Viral Pathogens:

“Viral pathogens” includes all plant viruses such as, for example,tobacco or cucumber mosaic virus, ringspot virus, necrosis virus, maizedwarf mosaic virus and the like.

The pathogens and diseases associated with them which are mentioned inTable 6 may be mentioned by way of example, but not by limitation. TABLE6 Viral diseases Disease Pathogen American wheat striate American wheatstriate mosaic virus (wheat striate mosaic) (AWSMV) Barley stripe mosaicBarley stripe mosaic virus (BSMV) Barley yellow dwarf Barley yellowdwarf virus (BYDV) Brome mosaic Brome mosaic virus (BMV) Cerealchlorotic mottle Cereal chlorotic mottle virus (CCMV) Corn chloroticvein Corn chlorotic vein banding virus banding (Brazilian maize (CCVBV)mosaic) Corn lethal necrosis Virus complex of Maize chlorotic mottlevirus (MCMV) and Maize dwarf mosaic virus (MDMV) A or B or Wheat streakmosaic virus(WSMV) Cucumber mosaic Cucumber mosaic virus (CMV) Cynodonchlorotic streak Cynodon chlorotic streak virus (CCSV) Johnsongrassmosaic Johnsongrass mosaic virus (JGMV) Maize bushy stuntMycoplasma-like organism (MLO) associated Maize chlorotic dwarf Maizechlorotic dwarf virus (MCDV) Maize chlorotic mottle Maize chloroticmottle virus (MCMV) Maize dwarf mosaic Maize dwarf mosaic virus (MDMV)strains A, D, E and F Maize leaf fleck Maize leaf fleck virus (MLFV)Maize line Maize line virus (MLV) Maize mosaic (corn leaf Maize mosaicvirus (MMV) stripe, enanismo rayado) Maize mottle and Maize mottle andchlorotic stunt virus chlorotic stunt Maize pellucid ringspot Maizepellucid ringspot virus (MPRV) Maize raya gruesa Maize raya gruesa virus(MRGV) Maize rayado fino (fine Maize rayado fino virus (MRFV) stripingdisease) Maize red leaf and red Mollicute stripe Maize red stripe Maizered stripe virus (MRSV) Maize ring mottle Maize ring mottle virus (MRMV)Maize rio IV Maize rio cuarto virus (MRCV) Maize rough dwarf Maize roughdwarf virus (MRDV) (Cereal (nanismo ruvido) tillering disease virus)Maize sterile stunt Maize sterile stunt virus (strains of barley yellowstriate virus) Maize streak Maize streak virus (MSV) Maize stripe (maizeMaize stripe virus chlorotic stripe, maize hoja blanca) Maize stuntingMaize stunting virus Maize tassel abortion Maize tassel abortion virus(MTAV) Maize vein enation Maize vein enation virus (MVEV) Maize wallabyear Maize wallaby ear virus (MWEV) Maize white leaf Maize white leafvirus Maize white line mosaic Maize white line mosaic virus (MWLMV)Millet red leaf Millet red leaf virus (MRLV) Northern cereal mosaicNorthern cereal mosaic virus (NCMV) Oat pseudorosette Oat pseudorosettevirus (zakuklivanie) Oat sterile dwarf Oat sterile dwarf virus (OSDV)Rice black-streaked Rice black-streaked dwarf virus dwarf (RBSDV) Ricestripe Rice stripe virus (RSV) Sorghum mosaic Sorghum mosaic virus(SrMV) (also: sugarcane mosaic virus (SCMV) strains H, I and M)Sugarcane Fiji disease Sugarcane Fiji disease virus (FDV) Sugarcanemosaic Sugarcane mosaic virus (SCMV) strains A, B, D, E, SC, BC, Sabiand MB (formerly MDMV-B) Wheat spot mosaic Wheat spot mosaic virus(WSMV)4. Animal Pests4.1 Insect Pathogens:

The following may be mentioned by way of example, but not by limitation:insects such as, for example, beetles, caterpillars, aphids or mites.Preferred insects are those of the genera Coleoptera, Diptera,Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera,Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera,etc. Especially preferred are coleopteran and lepidopteran insects suchas, for example, the European corn borer (ECB), Diabrotica barberi,Diabrotica undecimpunctata, Diabrotica virgifera, Agrotis ipsilon,Crymodes devastator, Feltia ducens, Agrotis gladiaria, Melanotus spp.,Aeolus mellillus, Aeolus mancus, Horistonotus uhlerii, Sphenophorusmaidis, Sphenophorus zeae, Sphenophorus parvulus, Sphenophorus callosus,Phyllogphaga spp., Anuraphis maidiradicis, Delia platura, Colaspisbrunnea, Stenolophus lecontei and Clivinia impressifrons.

Other examples are: lema (Oulema melanopus), frit fly (Oscinella frit),wireworms (Agrotis lineatus) and aphids (such as, for example, the oatgrain aphid Rhopalosiphum padi, the blackberry aphid Sitobion avenae).

4.2 Nematodes:

The pathogens and the diseases associated with them mentioned in Table 7may be mentioned by way of example, but not by limitation. TABLE 7Parasitic nematodes Damage Pathogenic nematode Awl Dolichodorus spp., D.heterocephalus Bulb and stem nematode, Ditylenchus dipsaci stem eelwormof rye Burrowing Radopholus similis Oat cyst nematode Heterodera avenae,H. zeae, Punctodera chalcoensis Dagger Xiphinema spp., X. americanum, X.mediterraneum False root-knot Nacobbus dorsalis Lance, ColumbiaHoplolaimus columbus Lance Hoplolaimus spp., H. galeatus LesionPratylenchus spp., P. brachyurus, P. crenatus, P. hexincisus, P.neglectus, P. penetrans, P. scribneri, P. thornei, P. zeae NeedleLongidorus spp., L. breviannulatus Ring Criconemella spp., C. ornataRoot-knot nematode Meloidogyne spp., M. chitwoodi, M. incognita, M.javanica Spiral Helicotylenchus spp. Sting Belonolaimus spp., B.longicaudatus Stubby-root Paratrichodorus spp., P. christiei, P. minor,Quinisulcius acutus, Trichodorus spp. Stunt Tylenchorhynchus dubius

Very especially preferred are Globodera rostochiensis and G. pallida(cyst eelworm on potato, tomato and other Solanaceae), Heteroderaschachtii (beet cyst eelworm on sugar and fodder beet, oilseed rape,cabbage and the like), Heterodera avenae (oat cyst nematode on oats andother cereal species), Ditylenchus dipsaci (stem or bulb eelworm, stemeelworm of rye, oats, maize, clover, tobacco, beet), Anguina tritici(grain nematode, cockle disease of wheat (spelt, rye), Meloidogyne hapla(root-knot nematode of carrot, cucumber, lettuce, tomato, potato, sugarbeet, lucerne).

Examples of preferred fungal or viral pathogens for the individualvarieties are:

1. Barley:

Fungal, bacterial and viral pathogens: Puccinia graminis f.sp. hordei,Blumeria (Erysiphe) graminis f.sp. hordei, barley yellow dwarf virus(BYDV),

Pathogenic insects/nematodes: Ostrinia nubilalis (European corn borer);Agrotis ipsilon; Schizaphis graminum; Blissus leucopterus leucopterus;Acrosternum hilare; Euschistus servus; Deliaplatura; Mayetioladestructor; Petrobia latens.

2. Soybean:

Fungal, bacterial or viral pathogens: Phytophthora megasperma fsp.glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotiniasclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae(Phomopsis sojae), Diaporthe phaseolorum var. caulivora, Sclerotiumrolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora manshurica,Colletotrichum dematium (Colletotrichum truncatum), Corynesporacassiicola, Septoria glycines, Phyllosticta sojicola, Alternariaalternata, Pseudomonas syringae p.v. glycinea, Xanthomonas campestrisp.v. phaseoli, Microsphaera diffussa, Fusarium semitectum, Phialophoragregata, soybean mosaic virus, Glomerella glycines, Tobacco Ring spotvirus, Tobacco Streak virus, Phakopsora pachyrhizi, Pythiumaphanidermatum, Pythium ultimum, Pythium debaryanum, Tomato spotted wiltvirus, Heterodera glycines, Fusarium solani.

Pathogenic insects/nematodes: Pseudoplusia includens; Anticarsiagemmatalis; Plathypena scabra; Ostrinia nubilalis; Agrotis ipsilon;Spodoptera exigua; Heliothis virescens; Helicoverpa zea; Epilachnavarivestis; Myzus persicae; Empoasca fabae; Acrosternum hilare;Melanoplus femurrubrum; Melanoplus differentialis; Hylemya platura;Sericothrips variabilis; Thrips tabaci; Tetranychus turkestani;Tetranychus urticae.

3. Canola:

Fungal, bacterial or viral pathogens: Albugo candida, Alternariabrassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotiniasclerotiorum, Mycosphaerella brassicola, Pythium ultimum, Peronosporaparasitica, Fusarium roseum, Alternaria alternata.

4. Alfalfa:

Fungal, bacterial or viral pathogens: Clavibater michiganese subsp.insidiosum, Pythium ultimum, Pythium irregulare, Pythium splendens,Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma,Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercosporamedicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis,Fusarium, Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches,Stemphylium herbarum, Stemphylium alfalfae.

5. Wheat:

Fungal, bacterial or viral pathogens: Pseudomonas syringae p.v.atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v.translucens, Pseudomonas syringae p.v. syringae, Alternaria alternata,Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum,Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporiumgramineum, Collotetrichum graminicola, Erysiphe graminis f.sp. tritici,Puccinia graminis f.sp. tritici, Puccinia recondita f.sp. tritici,Puccinia striiformis, Pyrenophora tritici-repentis, Septoria(Stagonospora) nodorum, Septoria tritici, Septoria avenae,Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctoniacerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum,Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana, BarleyYellow Dwarf Virus, Brome Mosaic Virus, soil borne wheat mosaic virus,wheat streak mosaic virus, wheat spindle streak virus, American wheatstriate virus, Claviceps purpurea, Tilletia tritici, Tilletia laevis,Ustilago tritici, Tilletia indica, Rhizoctonia solani, Pythiumarrhenomannes, Pythium gramicola, Pythium aphanidermatum, high plainsvirus, European wheat striate virus, Puccinia graminis f.sp. tritici(wheat stem rust), Blumeria (Erysiphe) graminis f.sp. tritici (wheatpowdery mildew).

Pathogenic insects/nematodes: Pseudaletia unipunctata; Spodoptera,frugiperda; Elasmopalpus lignosellus; Agrotis orthogonia; ElasmopalpusZignosellus; Oulema melanopus; Hypera punctata; Diabroticaundecimpunctata howardi; Russian wheat aphid; Schizaphis graminum;Macrosiphum avenae; Melanoplus femurrubrum; Melanoplus differentialis;Melanoplus sanguinipes; Mayetiola destructor; Sitodiplosis mosellana;Meromyza americana; Hylemya coarctata; Frankliniella fusca; Cephuscinctus; Aceria tulipae;

6. Sunflower:

Fungal, bacterial or viral pathogens: Plasmophora halstedii, Sclerotiniasclerotiorum, aster yellows, Septoria helianthi, Phomopsis helianthi,Alternaria helianthi, Alternaria zinniae, Botrytis cinerea, Phomamacdonaldii, Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopusoryzae, Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi,Verticillium dahliae, Erwinia carotovorum p.v. Carotovora,Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis.

Pathogenic insects/nematodes: Suleima helianthana; Homoeosomaelectellum; zygogramma exclamationis; Bothyrus gibbosus; Neolasiopteramurtfeldtiana.

7. Maize:

Fungal, bacterial or viral pathogens: Fusarium moniliforme var.subglutinans, Erwinia stewartii, Fusarium moniliforme, Gibberella zeae(Fusarium graminearum), Stenocarpella maydi (Diplodia maydis), Pythiumirregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens,Pythium ultimum, Pythium aphanidermatum, Aspergillus flavus, Bipolarismaydis 0, T (Cochliobolus heterostrophus), Helminthosporium carbonum I,II & III (Cochliobolus carbonum), Exserohilum turcicum I, II & III,Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis,Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi,Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum,Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvulariainaequalis, Curvularia pallescens, Clavibacter michiganese subsp.nebraskense, Trichoderma viride, Maize Dwarf Mosaic Virus A & B, WheatStreak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi,Pseudonomas avenae, Erwinia chrysanthemi p.v. Zea, Erwinia corotovora,Cornstunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora,Peronosclerospora sorghi, Peronosclerospora philippinesis,Peronosclerospora maydis, Peronosclerospora sacchari, Spacelothecareiliana, Physopella zeae, Cephalosporium maydis, Caphalosporiumacremonium, Maize Chlorotic Mottle Virus, High Plains Virus, MaizeMosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus (MSV,Maisstrichel-Virus), Maize Stripe Virus, Maize Rough Dwarf Virus.

Pathogenic insects/nematodes: Ostrinia nubilalis; Agrotis ipsilon;Helicoverpa zea; Spodoptera frugiperda; Diatraea grandiosella;Elasmopalpus lignosellus; Diatraea saccharalis; Diabrotica virgifera;Diabrotica longicornis barberi; Diabrotica undecimpunctata howardi;Melanotus spp.; Cyclocephala borealis; Cyclocephala immaculata; Popilliajaponica; Chaetocnema pulicaria; Sphenophorus maidis; Rhopalosiphummaidis; Anuraphis maidiradicis; Blissus leucopterus leucopterus;Melanoplus femurrubrum; Melanoplus sanguinipes; Hylemva platura;Agromyza parvicornis; Anaphothrips obscrurus; Solenopsis milesta;Tetranychus urticae.

8. Sorghum:

Fungal, bacterial or viral pathogens: Exserohilum turcicum,Colletotrichum graminicola (Glomerella graminicola), Cercospora sorghi,Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p.v.syringae, Xanthomonas campestris p.v. holcicola, Pseudomonasandropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconiacircinata, Fusarium moniliforme, Alternaria alternate, Bipolarissorghicola, Helminthosporium sorghicola, Curvularia lunata, Phomainsidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulisporasorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisoriumreilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisoriumsorghi, sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Clavicepssorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthonamacrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis,Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum,Pythium arrhenomanes, Pythium graminicola.

Pathogenic insects/nematodes: Chilo partellus; Spodoptera frugiperda;Helicoverpa zea; Elasmopalpus lignosellus; Feltia subterranea;Phyllophaga crinita; Eleodes, Conoderus and Aeolus spp.; Oulemamelanopus; Chaetocnema pulicaria; Sphenophorus maidis; Rhopalosiphummaidis; Sipha flava; Blissus leucopterus leucopterus; Contariniasorghicola; Tetranychus cinnabarinus; Tetranychus urticae.

9. Cotton:

Pathogenic insects/nematodes: Heliothis virescens; Helicoverpa zea;Spodoptera exigua; Pectinophora gossypiella; Anthonomus grandis grandis;Aphis gossypii; Pseudatomoscelis seriatus; Trialeurodes abutilonea;Lygus lineolaris; Melanoplus femurrubrum; Melanoplus differentialis;Thrips tabaci (onion thrips); Franklinkiella fusca; Tetranychuscinnabarinus; Tetranychus urticae.

10. Rice:

Pathogenic insects/nematodes: Diatraea saccharalis; Spodopterafrugiperda; Helicoverpa zea; Colaspis brunnea; Lissorhoptrusoryzophilus; Sitophilus oryzae; Nephotettix nigropictus; Blissusleucopterus; Acrosternum hilare.

11. Oilseed Rape:

Pathogenic insects/nematodes: Brevicoryne brassicae; Phyilotretacruciferae; Mamestra conjgurata; Plutella xylostella; Delia ssp.

For the purposes of the invention, “BI1 protein” is understood asmeaning polypeptides which have at least one sequence with at least 50%,preferably at least 80%, especially preferably at least 90%, veryespecially preferably 100% homology with a BI1 consensus motif selectedfrom the group consisting of a)  H(L/I)KXVY b)  AXGA(Y/F)XH c)  NIGG d) P(V/P) (Y/F)E(E/Q) (R/Q)KR e)  (E/Q)G(A/S)S(V/I)GPL f)  DP(S/G) (L/I)(I/L) g)  V(G/A)T(A/S) (L/I)AF(A/G)CF(S/T) h)  YL(Y/F)LGG, preferablyEYLYLGG i)  L(L/V)SS(G/W)L(S/T) (I/M)L(L/M)W j)  DTGX(I/V) (I/V)E.

Especially preferred in this context is the BI consensus motif f)YL(Y/F)LGG, very especially preferably (EYLYLGG). This motif ischaracteristic of plant BI1 proteins.

It is especially preferred that sequences with homology to at least 2 or3 of these motifs (a to g) occur in a BI1 protein, very especiallypreferably at least 4 or 5, most preferably all motifs a to j. FurtherBI1-typical sequence motifs can be derived by the skilled worker withoutdifficulty from the sequence alignment of BI1 proteins as shown in FIG.1 or 6.

Particularly preferable are BI proteins which are encoded by apolypeptide comprising at least one sequence selected from the groupconsisting of:

-   a) the sequences as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,    18, 20, 22, 24, 26, 28, 30, 32 and 38, and-   b) sequences which have at least 50%, preferably at least 70%,    especially preferably at least 90%, very especially preferably at    least 95% identity with one of the sequences as shown in SEQ ID NO:    2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 and 38,-   c) sequences which comprise at least one part-sequence of at least    10, preferably 20, especially preferably 50 contiguous amino acid    residues of one of the sequences as shown in SEQ ID NO: 2, 4, 6, 8,    10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 and 38.

In accordance with the invention, the term BI protein comprises inparticular natural or artificial mutations of the BI1 polypeptide asshown in SEQ ID NO: 2, 4, 6, 8, 10 and 38 and homologous polypeptidesfrom other organisms, preferably plants, which continue to haveessentially identical characteristics. Mutations comprise substitutions,additions, deletions, inversions or insertions of one or more amino acidresidues. This means that use forms using BI1 proteins from nonplantorganisms such as, for example, humans (GenBank Acc. No.: P55061), rats(GenBank Acc. No.: P55062) or Drosophila (GenBank Acc. No.: Q9VSH3) arealso comprised. Motifs which are conserved between plant and nonplantBI1 proteins can be identified easily by sequence alignment (cf.Alignment in Bolduc et 1. (2003) Planta 216:377-386; FIGS. 1 and 6).

Thus, polypeptides which are also comprised by the present invention arefor example those which are obtained by modification of a polypeptide asshown in SEQ ID NO: 2, 4, 6, 8, 10 and 38.

The sequences, from other plants, which are homologous to the BI1sequences disclosed within the scope of the present invention can beidentified for example by

-   a) database search in libraries of organisms whose genomic sequence    or cDNA sequence is known in its entirety or in part, using the BI1    sequences provided as search sequence or-   b) screening gene libraries or cDNA libraries using the BI1    sequences provided as probes.

Screening cDNA libraries or genomic libraries (for example using one ofthe nucleic acid sequences described in SEQ ID NO: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and 37 or parts of these asprobes) is a method, known to the skilled worker, for identifyinghomologs in other species. In this context, the probes derived from thenucleic acid sequences as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31 and 37 have a length of at least 20 bp,preferably at least 50 bp, especially preferably at least 100 bp, veryespecially preferably at least 200 bp, most preferably at least 200 bp.A DNA strand which is complementary to the sequences described as SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and 37 mayalso be used for screening the libraries.

Homology between two nucleic acid sequences is understood as meaning theidentity of the nucleic acid sequence over in each case the entiresequence length which is calculated by comparison with the aid of theprogram algorithm GAP (Wisconsin Package Version 10.0, University ofWisconsin, Genetics Computer Group (GCG), Madison, USA; Altschul et al.(1997) Nucleic Acids Res. 25:3389 et seq.), setting the followingparameters: Gap weight: 50 Length weight: 3 Average match: 10 Averagemismatch: 0

For example a sequence which has at least 80% homology with sequence SEQID NO: 1 at the nucleic acid level is understood as meaning a sequencewhich, upon comparison with the sequence SEQ ID NO: 1 by the aboveprogram algorithm with the above parameter set, has at least 80%homology.

Homology between two polypeptides is understood as meaning the identityof the amino acid sequence over in each case the entire sequence lengthwhich is calculated by comparison with the aid of the program algorithmGAP (Wisconsin Package Version 10.0, University of Wisconsin, GeneticsComputer Group (GCG), Madison, USA), setting the following parameters:Gap weight: 8 Length weight: 2 Average match: 2,912 Average mismatch:−2,003

For example a sequence which has at least 80% homology with sequence SEQID NO: 2 at the protein level is understood as meaning a sequence which,upon comparison with the sequence SEQ ID NO: 2 by the above programalgorithm with the above parameter set, has at least 80% homology.

BI1 proteins also comprise those polypeptides which are encoded bynucleic acid sequences which hybridize under standard conditions with aBI1 nucleic acid sequence described by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31 and 37, with the nucleic acidsequence complementary thereto or with parts of the above and which havethe same essential characteristics as the proteins described as SEQ IDNO: 2, 4, 6, 8, 10 and 38.

“Standard hybridization conditions” is to be understood in the broadsense and means stringent or else less stringent hybridizationconditions. Such hybridization conditions are described, inter alia, bySambrook J, Fritsch E F, Maniatis T et al., in Molecular Cloning (ALaboratory Manual), 2nd Edition, Cold Spring Harbor Laboratory Press,1989, pages 9.31-9.57) or in Current Protocols in Molecular Biology,John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the conditionsduring the wash step can be selected from the range of conditionsdelimited by low-stringency conditions (approximately 2×SSC at 50° C.)and high-stringency conditions (approximately 0.2×SSC at 50° C.,preferably at 65° C.) (20×SSC: 0.3M sodium citrate, 3M NaCl, pH 7.0). Inaddition, the temperature during the wash step can be raised fromlow-stringency conditions at room temperature, approximately 22° C., tohigher-stringency conditions at approximately 65° C. Both of theparameters, salt concentration and temperature, can be variedsimultaneously, or else one of the two parameters can be kept constantwhile only the other is varied. Denaturants, for example formamide orSDS, may also be employed during the hybridization. In the presence of50% formamide, hybridization is preferably effected at 42° C.

Referring to a BI protein, “ essential characteristics” means forexample one or more of the following characteristics:

-   a) Conferring or increasing the pathogen resistance to at least one    pathogen when increasing the amount of protein or function of said    BI protein in at least one tissue of the plant, said tissue being    other than the leaf epidermis.-   b) Absence of a spontaneously induced cell death when increasing the    amount of protein or the function of the said BI protein.-   c) The characteristic of significantly inhibiting the BAX-induced    apoptosis in the case of transient cotransfection of Bax and said    BI1 protein, for example in HEK293 cells. Suitable methods are    described (Bolduc N et al. (2003) Planta 216:377-386).-   d) The presence of five to seven putative transmembrane domains    within said BI1 protein.-   e) Preferential localization in cell membranes, in particular in the    nuclear membrane, the ER membrane and/or the thylakoid membrane.

In this context, the quantitative manifestation of said characteristicsof a BI1 protein can deviate in both directions in comparison with avalue obtained for the BI1 protein as shown in SEQ ID NO: 2, 4, 6, 8, 10or 38.

The term “increase of the amount or function of the BI1 protein” is tobe understood in the broad sense for the purposes of the presentinvention and may be the result of different cell-biological mechanisms.

“Amount of protein” means the amount of BI1 protein in the respectiveorganism, tissue, cell or cell compartment.

“Increase in the amount of protein” means the quantitative increase inthe amount of a BI1 protein in the respective organism, tissue, cell orcell compartment—for example by means of one of the methods describedhereinbelow—in comparison with the wild type of the same genus andspecies to which this method has not been applied, but on the otherwiseidentical overall conditions (such as, for example, culture conditions,age of the plants and the like). In this context, the increase amountsto at least 10%, preferably at least 30% or at least 50%, especiallypreferably at least 70% or 100%, very especially preferably by at least200% or 500%, most preferably by at least 1000%. The amount of proteincan be determined by a variety of methods with which the skilled workeris familiar. The following may be mentioned by way of example, but notby way of limitation: the micro-biuret method (Goa J (1953) Scand J ClinLab Invest 5:218-222), the Folin-Ciocalteu method (Lowry O H et al.(1951) J Biol Chem 193:265-275) or measuring the adsorption of CBB G-250(Bradford M M (1976) Analyt Biochem 72:248-254). Furthermore, it can bequantified by immunological methods such as, for example, Western blot.The preparation of suitable BI1 antibodies and the procedure for BI1Western blots are described (Bolduc et al. (2002) FEBS Lett532:111-114). Indirect quantification can be effected by a Northernblot, the amount of mRNA and the resulting amount of protein showing, asa rule, good correlation. Suitable methods are described (Bolduc et al.(2003) Planta 216:377-386; Matsumura H et al. (2003) Plant J 33:425-434,inter alia).

“Function” preferably means the characteristic of a BI1 protein ofreducing the spontaneously induced cell death and/or the characteristicof inhibiting the apoptosis-indicating effect of Bax. Such functionsbelong to the essential characteristic of a BI1 protein.

Within the context of the present invention, “increasing” the functionmeans, for example, the quantitative increase of the inhibitory effecton the Bax-induced apoptotic cell death, which can be quantified bymethods known to the skilled worker (see hereinabove). In this context,the increase amounts to at least 10%, preferably at least 30% or atleast 50%, very especially preferably at least 70% or 100%, veryespecially preferably by at least 200% or 500%, most preferably by atleast 1000%. Besides the above-described methods for increasing theamount of protein (which also always increases the function), methodsfor increasing the function comprise furthermore by way of example, butnot by limitation, in particular the introduction of mutations into aBI1 protein.

By way of example, but not by limitation, the amount of BI1 protein canbe increased by one of the following methods:

-   a) recombinant expression or overexpression of a BI1 protein by    introducing a recombinant expression cassette comprising a nucleic    acid sequence coding for a BI1 protein under the control of a    tissue-specific promoter, where said promoter has essentially no    activity in the leaf epidermis.-   b) modification (for example substitution) of the regulatory regions    (for example of the promoter region) of an endogenous BI1 gene, for    example substitution of a tissue-specific promoter by means of    homologous recombination, where said promoter has essentially no    activity in the leaf epidermis.-   c) Insertion of a nucleic acid sequence coding for a BI1 protein    into the plant genome downstream of a tissue-specific promoter by    means of homologous recombination, where said promoter has    essentially no activity in the leaf epidermis.-   d) increasing the expression of an endogenous BI1 protein by    introducing a transcription factor (for example an artificial    transcription factor from the class of the zinc finger proteins)    which is suitable for inducing the expression of said BI1 proteins.    It is preferred to introduce a recombinant expression cassette    comprising a nucleic acid sequence coding for said transcription    factor under the control of a tissue-specific promoter, where said    promoter has essentially no activity in the leaf epidermis.

For the purposes of the invention, the term “introduction” generallycomprises all those methods which are suitable for introducing, eitherdirectly or indirectly, the compound to be introduced into a plant orinto a cell, compartment, tissue, organ or seed thereof, or generatingit therein. Direct and indirect methods are comprised. The introductioncan lead to a transient presence of said compound or else to a stable orinducible presence. Introduction comprises methods such as, for example,transfection, transduction or transformation.

In the recombinant expression cassettes which are employed within theinvention, a nucleic acid molecule (for example coding for a BI1protein) is linked functionally to at least one tissue-specificpromoter, where said promoter has essentially no activity in the leafepidermis and where the promoter is heterologous with regard to thenucleic acid sequence to be expressed, i.e. does not naturally occur incombination with same. The recombinant expression cassettes according tothe invention can optionally comprise further genetic control elements.

Functional linkage is to be understood as meaning, for example, thesequential arrangement of said promoter with the nucleic acid sequenceto be expressed and, if appropriate, further regulatory elements suchas, for example, a terminator in such a way that each of the regulatoryelements can fulfil its function when the nucleic acid sequence isexpressed recombinantly. To this end, direct linkage in the chemicalsense is not necessarily required. Genetic control sequences such as,for example, enhancer sequences, can also exert their function on thetarget sequence from positions which are further away, or indeed fromother DNA molecules. Preferred arrangements are those in which thenucleic acid sequence to be expressed recombinantly is positioned behindthe sequence acting as promoter, so that the two sequences are linkedcovalently to each other. The distance between the promoter sequence andthe nucleic acid sequence to be expressed recombinantly is preferablyless than 200 base pairs, especially preferably less than 100 basepairs, very especially preferably less than 50 base pairs. A functionallinkage as well as a recombinant expression cassette can be generated bymeans of customary recombination and cloning techniques as aredescribed, for example, in Maniatis T, Fritsch E F and Sambrook J (1989)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor (NY), in Silhavy T J, Berman M L and Enquist L W(1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory,Cold Spring Harbor (NY), in Ausubel F M et al. (1987) Current Protocolsin Molecular Biology, Greene Publishing Assoc. and Wiley Interscienceand in Gelvin et al. (1990) In: Plant Molecular Biology Manual. However,further sequences which, for example, act as a linker with specificcleavage sites for restriction enzymes, or as a signal peptide, may alsobe positioned between the two sequences. The insertion of sequences mayalso lead to the expression of fusion proteins.

Preferably, the recombinant expression cassette, consisting of a linkageof promoter and nucleic acid sequence to be expressed, can exist in avector-integrated form and be inserted into a plant genome, for exampleby transformation.

However, recombinant expression cassette also denotes thoseconstructions in which the promoter is positioned in front of anendogenous BI1 gene, for example by means of homologous recombination,and thus controls the expression of the BI1 protein. Analogously, thenucleic acid sequence to be expressed (for example coding for a BI1protein) can also be positioned behind an endogenous promoter in such away that the same effect is manifested. Both approaches lead toinventive recombinant expression cassettes.

For the purposes of the present invention, a “tissue-specific promoterwhich has essentially no activity in the leaf epidermis” is generallyunderstood as meaning those promoters which are suitable for ensuring orincreasing a recombinant expression of a nucleic acid sequence at leastin one plant tissue, with the proviso that

-   a) said plant tissue is selected from among all plant tissues with    the exception of the leaf epidermis, and-   b) the recombinant expression under the control of said promoter in    said plant tissue amounts to at least five times, preferably at    least ten times, especially preferably at least one hundred times    the expression in the plant leaf epidermis.

The skilled worker is familiar with a number of promoters which meetthese requirements. Especially suitable are tissue-specific promoterssuch as, by way of example, but not by limitation, promoters withspecificity for the anthers, ovaries, flowers, stems, roots, tubers andseeds.

-   a) Preferred as seed-specific promoters are, for example, the    phaseolin promoter (U.S. Pat. No. 5,504,200; Bustos M M et al.    (1989). Plant Cell 1(9):839-53), the 2S albumin gene promoter    (Joseffson L G et al. (1987) J Biol Chem 262:12196-12201), the    legumin promoter (Shirsat A et al. (1989) Mol Gen Genet 215(2):    326-331) and the legumin B4 promoter (LeB4; Baumlein H et al. (1991)    Mol Gen Genet 225: 121-128; Baumlein H et al. (1992) Plant J    2(2):233-9; Fiedler U et al. (1995) Biotechnology (NY) 13(10):1090    seq.), the USP (unknown seed protein) promoter (Baumlein H et    al. (1991) Mol Gen Genet 225(3):459-67), the napin gene promoter    (U.S. Pat. No. 5,608,152; Stalberg K et al. (1996) L Planta    199:515-519), the sucrose binding protein promoter (WO 00/26388)    oleosin promoter (WO 98/45461), or the Brassica Bce4 promoter (WO    91/13980). Further suitable seed-specific promoters are those of the    genes encoding the high-molecular-weight glutenin (HMWG), gliadin,    branching enzyme, ADP glucose pyrophosphatase (AGPase) or starch    synthase. Furthermore preferred are promoters which permit    seed-specific expression in monocots such as maize, barley, wheat,    rye, rice and the like. The following can be employed    advantageously: the promoter of the lpt2 or lpt1 gene (WO 95/15389,    WO 95/23230) or the promoters described in WO 99/16890 (promoters of    the hordein gene, the glutelin gene, the oryzin gene, the prolamin    gene, the gliadin gene, the glutelin gene, the zein gene, the    kasirin gene or the secalin gene). Further seed-specific promoters    are described in WO 89/03887.-   b) Tuber-, storage-root- or root-specific promoters comprise, for    example, the promoter of the patatin gene (GenBank Acc. No.:    A08215), the patatin class I B33 promoter (GenBank Acc. No.: X14483)    or the cathepsin D inhibitor promoter from potato. Especially    preferred is the promoter described by SEQ ID NO: 29. Tuber-specific    promoters are especially suitable for achieving a resistance to    Phytophthora infestans in accordance with the invention. Since    obligate-biotrophic fungi only attack leaves, an activity in the    epidermal tuber tissue is irrelevant.-   c) Flower-specific promoters comprise, for example, the phytoene    synthase promoter (WO 92/16635) or the promoter of the P-rr gene (WO    98/22593).-   d) Anther-specific promoters comprise, for example, the 5126    promoter (U.S. Pat. No. 5,689,049, U.S. Pat. No. 5,689,051), the    glob-1 promoter and the γ-zein promoter.-   e) Ear-specific promoters such as, for example, the promoter in U.S.    Pat. No. 6,291,666. Ear-specific promoters are advantageous in    particular for mediating resistance to Fusarium.-   f) Mesophyll-specific promoters such as, for example, the promoter    of the wheat germin 9f-3.8 gene (GenBank Acc.-No.: M63224) or the    barley GerA promoter (WO 02/057412). Said promoters are particularly    advantageous since they are not only mesophyll-specific, but also    pathogen-inducible. Furthermore suitable are the mesophyll-specific    Arabidopsis CAB-2 promoter (GenBank Acc. No.: X15222), and the Zea    mays PPCZm1 promoter (GenBank Acc. No.: X63869). Particularly    preferred are the promoters described by SEQ ID NO: 30, 31 or 32.

The nucleic acid sequences present in the recombinant expressioncassettes or vectors according to the invention can be linkedfunctionally to further genetic control sequences in addition to apromoter. The term “genetic control sequences” is to be understood inthe broad sense and refers to all those sequences which have an effecton the materialization or the function of the recombinant expressioncassette according to the invention. Genetic control sequences alsocomprise further promoters, promoter elements or minimal promoters, allof which can modify the expression-governing properties. Thus, forexample, the tissue-specific expression may additionally depend oncertain stress factors, owing to genetic control sequences. Suchelements have been described, for example, for water stress, abscisicacid (Lam E and Chua N H, (1991) J Biol Chem; 266(26): 17131-17135) andheat stress (Schoffl F et al., (1989) Mol Gen Genet 217(2-3):246-53.

Genetic control sequences furthermore also comprise the 5′-untranslatedregions, introns or noncoding 3′-region of genes, such as, for example,the actin-1 intron, or the Adh1-S introns 1, 2 and 6 (general reference:The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer,New York (1994)). It has been demonstrated that they may play asignificant role in the regulation of gene expression. Thus, it has beendemonstrated that 5′-untranslated sequences can enhance the transientexpression of heterologous genes. Examples of translation enhancerswhich may be mentioned are the tobacco mosaic virus 5′ leader sequence(Gallie et al. (1987) Nucl Acids Res 15:8693-8711) and the like.Furthermore, they may promote tissue specificity (Rouster J et al.(1998) Plant J 15:435-440).

The recombinant expression cassette may advantageously comprise one ormore of what are known as enhancer sequences, linked functionally to thepromoter, which make possible an increased recombinant expression of thenucleic acid sequence. Additional advantageous sequences, such asfurther regulatory elements or terminators, may also be inserted at the3′ end of the nucleic acid sequences to be expressed recombinantly. Oneor more copies of the nucleic acid sequences to be expressedrecombinantly may be present in the gene construct.

Polyadenylation signals which are suitable as control sequences areplant polyadenylation signals, preferably those which essentiallycorrespond to T-DNA polyadenylation signals from Agrobacteriumtumefaciens, in particular the OCS (octopin synthase) terminator and theNOS (nopalin synthase) terminator.

Control sequences are furthermore to be understood as those which makepossible homologous recombination or insertion into the genome of a hostorganism or which permit removal from the genome. In the case ofhomologous recombination, for example the natural promoter of a BI1 genemay be exchanged for one of the preferred tissue-specific promoters.Methods such as the cre/lox technology permit a tissue-specific, ifappropriate inducible, removal of the recombinant expression cassettefrom the genome of the host organism (Sauer B (1998) Methods.14(4):381-92). In this method, specific flanking sequences (loxsequences), which later allow removal by means of cre recombinase, areattached to the target gene.

A recombinant expression cassette and the vectors derived from it maycomprise further functional elements. The term functional element is tobe understood in the broad sense and refers to all those elements whichhave an effect on the generation, amplification or function of therecombinant expression cassettes, vectors or recombinant organismsaccording to the invention. The following may be mentioned by way ofexample, but not by limitation:

-   a) Selection markers which confer resistance to a metabolism    inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456),    antibiotics or biocides, preferably herbicides, such as, for    example, kanamycin, G 418, bleomycin or hygromycin, or else    phosphinothricin and the like. Especially preferred selection    markers are those which confer resistance to herbicides. Examples    which may be mentioned are: DNA sequences which encode    phosphinothricin acetyl transferases (PAT) and which inactivate    glutamin synthase inhibitors (bar and pat genes),    5-enolpyruvylshikimate-3-phosphate synthase genes (EPSP synthase    genes), which confer resistance to    Glyphosat®(N-(phosphonomethyl)glycine), the gox gene, which codes    for Glyphosat®-degrading enzymes (Glyphosate oxidoreductase), the    deh gene (encoding a dehalogenase which inactivates dalapon®),    sulfonylurea- and imidazolinone-inactivating acetolactate synthases,    and bxn genes, which encode bromoxynil-degrading nitrilase enzymes,    the aasa gene, which confers resistance to the antibiotic    apectinomycin, the streptomycin phosphotransferase (SPT) gene, which    allows resistance to streptomycin, the neomycin phosphotransferase    (NPTII) gene, which confers resistance to kanamycin or geneticidin,    the hygromycin phosphotransferase (HPT) gene, which mediates    resistance to hygromycin, the acetolactate synthase gene (ALS),    which confers resistance to sulfonylurea herbicides (for example    mutated ALS variants with, for example, the S4 and/or Hra mutation).-   b) Reporter genes which encode readily quantifiable proteins and,    via their color or enzyme activity, make possible an assessment of    the transformation efficacy, the site of expression or the time of    expression. Very especially preferred in this context are reporter    proteins (Schenborn E, Groskreutz D. Mol Biotechnol. 1999;    13(1):29-44) such as the green fluorescent protein (GFP) (Sheen et    al. (1995) Plant Journal 8(5):777-784; Haseloff et al. (1997) Proc    Natl Acad Sci USA 94(6):2122-2127; Reichel et al. (1996) Proc Natl    Acad Sci USA 93(12):5888-5893; Tian et al. (1997) Plant Cell Rep    16:267-271; WO 97/41228; Chui W L et al. (1996) Curr Biol 6:325-330;    Leffel S M et al. (1997) Biotechniques. 23(5):912-8),    chloramphenicol transferase, a luciferase (Ow et al. (1986) Science    234:856-859; Millar et al. (1992) Plant Mol Biol Rep 10:324-414),    the aequorin gene (Prasher et al. (1985) Biochem Biophys Res Commun    126(3):1259-1268), β-galactosidase, R locus gene (encoding a protein    which regulates the production of anthocyanin pigments (red    coloring) in plant tissue and thus makes possible a direct analysis    of the promoter activity without addition of further auxiliary    substances or chromogenic substrates; Dellaporta et al., In:    Chromosome Structure and Function: Impact of New Concepts, 18th    Stadler Genetics Symposium, 11:263-282, 1988), with β-glucuronidase    being very especially preferred (Jefferson et al., EMBO J. 1987, 6,    3901-3907).-   c) Origins of replication, which ensure amplification of the    recombinant expression cassettes or vectors according to the    invention in, for example, E. coli. Examples which may be mentioned    are ORI (origin of DNA replication), the pBR322 ori or the P15A ori    (Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed.    Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,    1989).-   d) Elements which are necessary for Agrobacterium-mediated plant    transformation, such as, for example, the right or left border of    the T-DNA or the vir region.

To select cells which have successfully undergone homologousrecombination, or else to select transformed cells, it is, as a rule,necessary additionally to introduce a selectable marker, which confersresistance to a biocide (for example herbicide), a metabolism inhibitorsuch as 2-deoxyglucose-6-phosphate (WO 98/45456) or an antibiotic to thecells which have successfully undergone recombination. The selectionmarker permits the selection of the transformed cells from untransformedones (McCormick et al. (1986) Plant Cell Reports 5:81-84).

The introduction of a recombinant expression cassette according to theinvention into an organism or cells, tissues, organs, parts or seedsthereof (preferably into plants or plant cells, tissue, organs, parts orseeds) can be effected advantageously using vectors which comprise therecombinant expression cassettes. The recombinant expression cassettecan be introduced into the vector (for example a plasmid) via a suitablerestriction cleavage site. The plasmid formed is first introduced intoE. coli. Correctly transformed E. coli are selected, grown, and therecombinant plasmid is obtained by the methods familiar to the skilledworker. Restriction analysis and sequencing may serve to verify thecloning step.

Examples of vectors may be plasmids, cosmids, phages, viruses or elseagrobacteria. In an advantageous embodiment, the recombinant expressioncassette is introduced by means of plasmid vectors. Preferred vectorsare those which make possible stable integration of the recombinantexpression cassette into the host genome.

The generation of a transformed organism (or of a transformed cell ortissue) requires introducing the DNA, RNA or protein in question intothe relevant host cell.

A multiplicity of methods are available for this procedure, which istermed transformation (or transduction or transfection) (Keown et al.(1990) Methods Enzymol 185:527-537); Jenes B et al. (1993) Techniquesfor Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering andUtilization, published by SD Kung and R Wu, Academic Press, P. 128-143and in in Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol42:205-225).

For example, the DNA or RNA can be introduced directly by microinjectionor by bombardment with DNA-coated microparticles. Also, the cell can bepermeabilized chemically, for example using polyethylene glycol, so thatDNA can enter the cell by diffusion. The DNA can also be introduced byprotoplast fusion with other DNA-containing units such as minicells,cells, lysosomes or liposomes. Another suitable method of introducingDNA is electroporation, where the cells are permeabilized reversibly byan electrical pulse. Suitable methods have been described (for exampleby Bilang et al. (1991) Gene 100:247-250; Scheid et al. (1991) Mol GenGenet 228:104-112; Guerche et al. (1987) Plant Science 52:111-116;Neuhause et al. (1987) Theor Appl Genet 75:30-36; Klein et al. (1987)Nature 327:70-73; Howell et al. (1980) Science 208:1265; Horsch et al.(1985) Science 227:1229-1231; DeBlock et al. (1989) Plant Physiol91:694-701).

In plants, the above-described methods of transforming and regeneratingplants from plant tissues or plant cells are exploited for transient orstable transformation. Suitable methods are especially protoplasttransformation by polyethylene-glycol-induced DNA uptake, the biolisticmethod with the gene gun, what is known as the particle bombardmentmethod, electroporation, incubation of dry embryos in DNA-containingsolution, and microinjection.

In addition to these “direct” transformation techniques, transformationcan also be effected by bacterial infection by means of Agrobacteriumtumefaciens or Agrobacterium rhizogenes. The Agrobacterium-mediatedtransformation is best suited to dicotyledonous plant cells. The methodsare described, for example, by Horsch R B et al. (1985) Science 225:1229f).

When agrobacteria are used, the recombinant expression cassette must beintegrated into specific plasmids, either into a shuttle or intermediatevector, or into a binary vector. If a Ti or Ri plasmid is to be used forthe transformation, at least the right border, but in most cases theright and left border, of the Ti or Ri plasmid T-DNA is linked to therecombinant expression cassette to be introduced in the form of aflanking region.

Binary vectors are preferably used. Binary vectors are capable ofreplication both in E. coli and in Agrobacterium. As a rule, theycomprise a selection marker gene and a linker or polylinker flanked bythe right and left T-DNA border sequence. They can be transformeddirectly into Agrobacterium (Holsters et al. (1978) Mol Gen Genet163:181-187). The selection marker gene permits a selection oftransformed agrobacteria and is, for example, the nptII gene, whichconfers resistance to kanamycin. The Agrobacterium which acts as hostorganism in this case should already comprise a plasmid with the virregion. The latter is required for transferring the T-DNA to the plantcell. An Agrobacterium transformed in this way can be used fortransforming plant cells. The use of T-DNA for transforming plant cellshas been studied and described intensively (EP 120 516; Hoekema, In: TheBinary Plant Vector System, Offsetdrukkerij Kanters B. V., Alblasserdam,Chapter V; An et al. (1985) EMBO J 4:277-287). Various binary vectorsare known and some commercially available such as, for example, pBI101.2or pBIN19 (Bevan et al. (1984) Nucl Acids Res 12:8711f; ClontechLaboratories, Inc. USA). Further promoters suitable for expression inplants have been described (Rogers et al. (1987) Methods Enzymol153:253-277; Schardl et al. (1987) Gene 61:1-11; Berger et al. (1989)Proc Natl Acad Sci USA 86:8402-8406).

Direct transformation techniques are suitable in principle for anyorganism and cell type. The plasmid used need not meet any particularrequirements in the case of the injection or electroporation of DNA orRNA into plant cells. Simple plasmids such as those of the pUC seriescan be used. If complete plants are to be regenerated from thetransformed cells, it is necessary for an additional selectable markergene to be located on the plasmid.

Stably transformed cells, i.e. those which contain the introduced DNAintegrated into the DNA of the host cell, can be selected fromuntransformed cells when a selectable marker is part of the DNAintroduced. Examples of genes which can act as markers are all thosewhich are capable of conferring resistance to antibiotics or herbicides(such as kanamycin, G 418, bleomycin, hygromycin or phosphinothricin)(see above). Transformed cells which express such marker genes arecapable of surviving in the presence of concentrations of acorresponding antibiotic or herbicide which kill an untransformed wildtype. Examples of suitable selection markers are mentioned above. Once atransformed plant cell has been generated, a complete plant can beobtained using methods known to the skilled worker. For example, calluscultures are used as starting material. The development of shoot androot can be induced in this as yet undifferentiated cell biomass in aknown fashion. The plantlets obtained can be planted out and bred. Theskilled worker is familiar with methods of regenerating plant parts andintact plants from plant cells. Methods to do so are described, forexample, by Fennell et al. (1992) Plant Cell Rep. 11: 567-570; Stoegeret al (1995) Plant Cell Rep. 14:273-278; Jahne et al. (1994) Theor ApplGenet 89:525-533. The resulting plants can be bred and hybridized in thecustomary fashion. Two or more generations should be grown in order toensure that the genomic integration is stable and hereditary.

The method according to the invention can advantageously be combinedwith further methods which bring about pathogen resistance (for exampleto insects, fungi, bacteria, nematodes and the like), stress resistanceor another improvement of the plant properties. Examples are mentioned,inter alia, by Dunwell J M (2000), J Exp Bot. 51 Spec No: 487-96.

The invention furthermore relates to polypeptide sequences coding for aBI1 protein comprising at least one sequence selected from the groupconsisting of

-   a) the sequences as shown in SEQ ID NO: 12, 14, 16, 18, 20, 22, 24,    28, 30, 32 or 38,-   b) sequences which have least 90%, preferably at least 95%,    especially preferably at least 98%, homology with one of the    sequences as shown in SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 28, 30,    32 or 38, and-   c) sequences which comprise at least 10, preferably at least 20,    especially preferably at least 30, contiguous amino acids of one of    the sequences as shown in SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 28,    30, 32 or 38.

The invention furthermore relates to nucleic acid sequences coding forthe novel polypeptide sequences according to the invention which codefor BI1 proteins. Preferred are the nucleic acid sequence as shown inSEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 37, the nucleicacid sequence which is complementary thereto and the sequences derivedtherefrom as the result of degeneration of the genetic code.

The invention furthermore relates to recombinant expression cassetteswhich comprise one of the nucleic acid sequences according to theinvention. In the recombinant expression cassettes according to theinvention, the nucleic acid sequence encoding the barley BI1 protein islinked to at least one genetic control element as defined above in sucha manner that it is capable of expression (transcription and, ifappropriate, translation) in any organism, preferably in plants.Suitable genetic control elements are described above. The recombinantexpression cassettes may also comprise further functional elements inaccordance with the above definition. The inserted nucleic acid sequenceencoding a barley BI1 protein can be inserted in the expression cassettein sense or antisense orientation and thus lead to the expression ofsense or antisense RNA. Recombinant vectors comprising the recombinantexpression cassettes are also in accordance with the invention.

“Recombinant”, for example regarding a nucleic acid sequence, anexpression cassette or a vector comprising said nucleic acid sequence oran organism transformed with said nucleic acid sequence, expressioncassette or vector, refers to all those constructs originating bygenetic engineering methods in which either

-   a) the BI1 nucleic acid sequence, or-   b) a genetic control sequence linked functionally to the BI1 nucleic    acid sequence, for example a promoter, or-   c) (a) and (b)    are not located in their natural genetic environment or have been    modified by recombinant methods, an example of a modification being    substitutions, additions, deletions, inversions or insertions of one    or more nucleotide residues. Natural genetic environment refers to    the natural chromosomal locus in the organism of origin, or to the    presence in a genomic library. In the case of a genomic library, the    natural genetic environment of the nucleic acid sequence is    preferably retained, at least in part. The environment flanks the    nucleic acid sequence at least at one side and has a sequence of at    least 50 bp, preferably at least 500 bp, especially preferably at    least 1000 bp, very especially preferably at least 5000 bp, in    length. A naturally occurring expression cassette—for example the    naturally occurring combination of the BI1 promoter with the    corresponding BI1 gene—becomes a recombinant expression cassette    when it is modified by non-natural, synthetic (“artificial”) methods    such as, for example, mutagenization.

Such methods have been described (U.S. Pat. No. 5,565,350; WO 00/15815;also see above).

The invention also relates to recombinant organisms transformed with atleast one of the nucleic acid sequences according to the invention,expression cassette according to the invention or vector according tothe invention, and to cells, cell cultures, tissues, parts—such as, forexample, leaves, roots and the like in the case of plant organisms—orpropagation material derived from such organisms. The term organism isto be understood in the broad sense and refers to prokaryotic andeukaryotic organisms, preferably bacteria, yeasts, fungi, animalorganisms and plant organisms. Host organisms, or starting organisms,which are preferred as recombinant organisms are in particular plants asdefined above.

The invention furthermore relates to the use of the recombinantorganisms according to the invention and of the cells, cell cultures,parts—such as, for example, roots, leaves and the like in the case ofrecombinant plant organisms—derived from them, and to recombinantpropagation material such as seeds or fruits, for the production offoodstuffs or feeding stuffs, pharmaceuticals or fine chemicals.

Furthermore a nucleic acid molecule which is antisense to the nucleicacid according to the invention, is a monoclonal antibody which bindsspecifically to the polypeptide according to the invention and afungicide which comprises the nucleic acid according to the invention,the vector according to the invention, in particular an infectious, forexample viral, vector according to the invention, the polypeptideaccording to the invention in a form which is suitable for applicationto plants, for example in encapsulated form or in an infectious organismpreferably suitable for transferring nucleic acids or expressing genesin a cell, such as an Agrobacterium or a virus.

In one embodiment, the invention relates to the use of a nucleic acidmolecule which codes for BI-1, or of a BI-1 protein, for the generationof a pathogen-resistant plant, preferably for the generation of a plantwhich is resistant to fungi or for the generation of a fungicidebringing about the same, or for controlling or treating plants which areattacked, or liable to attack, by pathogens.

Sequences

-   1. SEQ ID NO: 1: Nucleic acid sequence coding for a BI1 protein from    barley (Hordeum vulgare).-   2. SEQ ID NO: 2: Amino acid sequence coding for a BI1 protein from    barley (Hordeum vulgare).-   3. SEQ ID NO: 3: Nucleic acid sequence coding for a BI1 protein from    Arabidopsis thaliana.-   4. SEQ ID NO: 4: Amino acid sequence coding for a BI1 protein from    Arabidopsis thaliana.-   5. SEQ ID NO: 5: Nucleic acid sequence coding for a BI1 protein from    tobacco.-   6. SEQ ID NO: 6: Amino acid sequence coding for a BI1 protein from    tobacco.-   7. SEQ ID NO: 7: Nucleic acid sequence coding for a BI1 protein from    rice.-   8. SEQ ID NO: 8: Amino acid sequence coding for a BI1 protein from    rice.-   9. SEQ ID NO: 9: Nucleic acid sequence coding for a BI1 protein from    oilseed rape.-   10. SEQ ID NO: 10: Amino acid sequence coding for a BI1 protein from    oilseed rape.-   11. SEQ ID NO: 11: Nucleic acid sequence coding for a part of a BI1    protein from soybean.-   12. SEQ ID NO: 12: Amino acid sequence coding for a part of a BI1    protein from soybean.-   13. SEQ ID NO: 13: Nucleic acid sequence coding for a part of a BI1    protein from soybean.-   14. SEQ ID NO: 14: Amino acid sequence coding for a part of a BI1    protein from soybean.-   15. SEQ ID NO: 15: Nucleic acid sequence coding for a part of a BI1    protein from wheat.-   16. SEQ ID NO: 16: Amino acid sequence coding for a part of a BI1    protein from wheat.-   17. SEQ ID NO: 17: Nucleic acid sequence coding for a part of a BI1    protein from maize.-   18. SEQ ID NO: 18: Amino acid sequence coding for a part of a BI1    protein from maize.-   19. SEQ ID NO: 19: Nucleic acid sequence coding for a part of a BI1    protein from wheat.-   20. SEQ ID NO: 20: Amino acid sequence coding for a part of a BI1    protein from wheat.-   21. SEQ ID NO: 21: Nucleic acid sequence coding for a part of a BI1    protein from maize.-   22. SEQ ID NO: 22: Amino acid sequence coding for a part of a BI1    protein from maize.-   23. SEQ ID NO: 23: Nucleic acid sequence coding for a part of a BI1    protein from maize.-   24. SEQ ID NO: 24: Amino acid sequence coding for a part of a BI1    protein from maize.-   25. SEQ ID NO: 25: Nucleic acid sequence coding for a part of a BI1    protein from wheat.-   26. SEQ ID NO: 26: Amino acid sequence coding for a part of a BI1    protein from wheat.-   27. SEQ ID NO: 27: Nucleic acid sequence coding for a part of a BI1    protein from maize.-   28. SEQ ID NO: 28: Amino acid sequence coding for a part of a BI1    protein from maize.-   29. SEQ ID NO: 29: Nucleic acid sequence coding for the patatin    promoter from potato.-   30. SEQ ID NO: 30: Nucleic acid sequence coding for the germin    9f-3.8 promoter from wheat.-   31. SEQ ID NO: 31: Nucleic acid sequence coding for the Arabidopsis    CAB-2 promoter.-   32. SEQ ID NO: 32: Nucleic acid sequence coding for the PPCZm1    promoter from maize.-   33. SEQ ID NO: 33: Nucleic acid sequence coding for the recombinant    expression vector pUbiBI-1.-   34. SEQ ID NO: 34: Nucleic acid sequence coding for the recombinant    expression vector pLo114UbiBI-1.-   35. SEQ ID NO: 35: Nucleic acid sequence coding for the recombinant    expression vector pOXoBI-1.-   36. SEQ ID NO: 36: Nucleic acid sequence coding for the recombinant    expression vector pLo114OXoBI-1.-   37. SEQ ID NO: 37: Nucleic acid sequence coding for BI-1 protein    from wheat.-   38. SEQ ID NO: 38: Amino acid sequence coding for a BI1 protein from    wheat.-   39. SEQ ID NO: 39: Nucleic acid sequence for PEN1 (=ROR2) from    barley.-   40. SEQ ID NO: 40: Amino acid sequence coding for PEN1 (=ROR2) from    barley.-   41. SEQ ID NO: 41: Nucleic acid sequence for PEN1 (=ROR2) from    Arabidopsis thaliana.-   42. SEQ ID NO: 42: Amino acid sequence coding for PEN1 (=ROR2) from    Arabidopsis thaliana.-   43. SEQ ID NO: 43: Nucleic acid sequence coding for SNAP34 from    barley.-   44. SEQ ID NO: 44: Amino acid sequence coding for SNAP34 from    barley.

FIGURES

1. FIG. 1 a-d: Alignment of protein sequences of different BI-1 proteinsfrom plants. AtBI-1: Arabidopsis; BnBI-1: Brassica napus (oilseed rape);GmBI2: Glycine max (soybean; variant 1); GmBI3: Glycine max (soybean;variant 2); HVBI-1: Hordeum vulgare (barley); NtBI-1: Nicotiana tabacum(tobacco); OsBI-1: Oryza sativa (rice); TaBI11: Triticum aestivum(wheat, variant 1); TaBI18: Triticum aestivum (wheat, variant 2); TaBI5new: Triticum aestivum (wheat, variant 3); ZmBI14: Zea mays (maize;variant 1); ZmBI16: Zea mays (maize; variant 2); ZmBI33: Zea mays(maize; variant 3); ZmBI8: Zea mays (maize; variant 4); Consensus:consensus sequence derived from the alignment.

2. FIG. 2: Vector map for the vector pUbiBI-1 (Ubi: ubiquitin promoter;BI-1 nucleic acid sequence coding for barley BI1 protein; ter:transcription terminator). Also shown are the localizations of thecleavage sites for different restriction enzymes.

3. FIG. 3: Vector map for the vector pLO114UbiBI-1 (Ubi: ubiquitinpromoter; BI-1 nucleic acid sequence coding for barley BI1 protein; ter:transcription terminator). Also shown are the localizations of thecleavage sites for different restriction enzymes.

4. FIG. 4: Vector map for the vector pOxoBI-1 (Oxo: TaGermin 9f-2.8promoter; BI-1 nucleic acid sequence coding for barley BI1 protein; ter:transcription terminator). Also shown are the localizations of thecleavage sites for different restriction enzymes.

5. FIG. 5: Vector map for the vector pLO114OxoBI-1 (Oxo: TaGermin 9f-2.8promoter; BI-1 nucleic acid sequence coding for barley BI1 protein; ter:transcription terminator). Also shown are the localizations of thecleavage sites for different restriction enzymes.

6. FIG. 6: Alignment of the protein sequences of BI-1 proteins frombarley (Hordeum vulgare, GenBank Acc. No.: CAC37797), rice (Oryzasativa, GenBank Acc. No.: Q9MBD8), Arabidopsis thaliana (GenBank Acc.No.: Q9LD45) and humans (Homo sapiens, GenBank Acc. No.: AAB87479).Amino acids shown against the black background are identical in allspecies. Amino acids shown against the gray background are identical inplants only. Bars indicate the predicted seven transmembrane domains inHvBI-1.

7. FIG. 7: BI-1 expression in resistant and susceptible barley lines(cDNA gel blot analysis): cDNAs were synthesized by means of RT-PCR,starting from total RNA. Total RNA was obtained from the susceptiblebarley line Pallas, the resistant barley line BCPM1a12 and the resistantbarley line BCPm1o5 at times 0 (i.e. immediately prior to inoculation)and in each case 1, 4 and 7 days after inoculation with Bgh and, inparallel, from uninfected control plants (Ø). The RT-PCR for BI-1 wascarried out using 20 cycles (see hereinbelow). The amount of RNAemployed (0.5 μg) was additionally checked in gels by means of rRNAstaining with ethidium bromide. A repetition of the experiments gavecomparable results.

8. FIG. 8: BI-1 is expressed in mesophyll tissue (cDNA gel blotanalysis). RT-PCR was carried out starting from RNA isolated from Pallas(P) and BCPM1a12 (P10) (24 h after inoculation with BghA6). To extractthe total RNA, abaxial epidermal strips (E, inoculated positions of theleaves) were separated from the mesophyll and the adaxial epidermis (M).Ubiquitin 1 (Ubi) was used as label for tissue-unspecific geneexpression. RT-PCR was carried out using 30 cycles.

9. FIG. 9: BI-1 expression is repressed during chemical resistanceinduction.

(A) Chemical induced resistance in the barley line Pallas gg. Blumeriagraminis (DC) Speer f.sp. hordei (Bgh). Barley primary leaves weretreated with 2,6-dichloroisonicotinic acid (DCINA) and showed fewermildew pustules than corresponding untreated control plants.

(B) RNA and cDNA Blots. RNA (10 μg) was analyzed 0, 1, 2 and 3 daysafter soil treatment (soil drench treatment; dpt) with DCINA and withthe control (carrier substance) and additionally 1 and 4 dayspost-inoculation (dpi, corresponds to 4 and 7 dpt, respectively). RT-PCR(Ubi, BI-1) was carried out using 20 cycles. Repetition resulted incomparable results (see Example 2).

BCI-4 was employed as the control. BCI-4 is a DCINA-induced gene (Besseret al. (2000) Mol Plant Pahol. 1(5): 277-286) and a member of the BarleyChemically (=BTH) induced gene family.

10. FIG. 10: Overexpression of BI-1 induced supersusceptibility.

(A) Mean penetration efficiency of Bgh in 6 independent experiments withBgh on barley line Ingrid. The PE of Bgh was significantly increased(p<0.01, Student's t-test) in cells which were transformed with pBI-1(by bombardment) in comparison with cells which were bombarded with theblank vector control (pGY1).

(B) The penetration efficiency of Bgh on cells which had been bombardedwith an antisense BI-1 construct (pasBI-1) was not significantly reduced(p>0.05) in comparison with cells which had been bombarded with theblank vector control (pGY1).

The columns show in each case the mean value of the individualexperiments. The bars represent the standard error.

11. FIG. 11: Overexpression of BI-1 induced breaking of them1o5-mediated penetration resistance.

The penetration efficiency of Bgh was assessed in 3 to 4 independentexperiments using Bgh on the barley lines Ingrid-m1o5 and pallas-m1o5.The PE caused by Bgh was significantly increased (p<0.05) in cells whichhad been transformed with pBI-1 (bombarded) in comparison with cellswhich had been bombarded with the blank vector control (pGY1). Thecolumns show in each case the mean value of three independentexperiments. The bars represent the standard error.

12. FIG. 12: The expression of BI-1 is induced by toxic culturefiltrates from Bipolaris sorokiniana. Northern blots (10 μg total RNA)with RNA from Ingrid (I) and BCIngrid-m1o5 (122). RNA was isolated 0,24, 48 and 72 hours after injection of the toxic culture filtrates ofBipolaris sorokiniana (T) or water (W). BI-1 mRNAs were detected onnylon membranes following stringent washing. BI-1: detection of BAXInhibitor 1 mRNA; Ubi: detection of Ubiquitin 1; Asprot: detection ofthe aspartate protease mRNA; hat: hours after treatment (“h aftertreatment”).

13. FIG. 13: BI-1 overexpression breaks non-host resistance of barley(cv. Manchuria) to Blumeria graminis f.sp. tritici. The penetrationrates were analyzed in three independent experiments.

EXAMPLES

General methods:

The chemical synthesis of oligonucleotides can be effected, for example,in the known fashion using the phosphoamidite method (Voet, Voet, 2ndEdition, Wiley Press New York, pages 896-897). The cloning steps carriedout for the purposes of the present invention such as, for example,restriction cleavages, agarose gel electrophoresis, purification of DNAfragments, transfer of nucleic acids to nitrocellulose and nylonmembranes, linking DNA fragments, transformation of E. coli cells,growing bacteria, multiplying phages and sequence analysis ofrecombinant DNA, are carried out as described by Sambrook et al. (1989)Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. The sequencingof recombinant DNA molecules is carried out with an MWG-Licor laserfluorescence DNA sequencer following the method of Sanger (Sanger et al.(1977). Proc Natl Acad Sci USA 74:5463-5467).

Example 1 Plants, Pathogens and Inoculation

The barley varieties Ingrid, Pallas and the backcrossed line BCPM1a12,BCPm1o5 and BCIngrid-m1o5 (I22) were donated by Lisa Munk, Department ofPlant Pathology, Royal Veterinary and Agricultural University,Copenhagen, Denmark. Its production has been described (Kølster P et al.(1986) Crop Sci 26: 903-907).

Unless otherwise specified, the seed which had been pregerminated onmoist filter paper for 12 to 36 hours in the dark was sown along theedge of a square pot (8×8 cm; 5 kernels per pot) in Fruhstorfer soil,type P, covered with soil and watered regularly with tap water. All ofthe plants were cultured in controlled-environment cabinets or chambersfor 5 to 8 days at 18° C., 60% relative atmospheric humidity and a16-hr-light/8-hr-dark rhythm at 3000 and 5000 lux, respectively (photonflow density 50 and 60 μmols-¹m-^(2·); respectively) and used in theexperiments during the seedling stage. In experiments in whichapplications to the primary leaves were carried out, the latter weredeveloped fully.

Before the transient transfection experiments were carried out, theplants were grown in controlled-environment cabinets or chambers at adaytime temperature of 24° C., a nighttime temperature of 20° C., 50 to60% relative atmospheric humidity and a 16-hr-light/8-hr-dark rhythm at30 000 lux.

Barley powdery mildew Blumeria graminis (DC) Speer f.sp. hordei Em.Marchal race A6 (Wiberg A (1974) Hereditas 77: 89-148) (BghA6) was usedfor the inoculation of barley plants. The fungus was provided by theDepartment of Biometry, JLU Gieβen. Inoculum was maintained incontrolled-environment cabinets under identical conditions to thosedescribed above for the plants by transferring the conidia of infectedplant material at a rate of 100 conidia/mm² to 7-day-old barley plantscv. Golden Promise, which were grown regularly.

Inoculation was carried out on primary leaves of barley plants with thefollowing conidial densities: 5 conidia/mm² in the case of chemicalresistance induction and macroscopic evaluation of the inductionsuccess, 50 conidia/mm² in the case of gene expression studies and 150conidia/mm² for the verification of the gene transformation usingtransformed leaf segments. The inoculation with BghA6 was carried outusing 7-day-old seedlings by shaking off the conidia of already infectedplants in an inoculation tower (unless otherwise specified).

Example 2 Modulation of the Expression of BI1 using DCINA

2,6-Dichloroisonicotinic acid (DCINA, Syngenta A G, Basle, Switzerland;as a 25% (w/w) formulation) was applied to 4-day-old barley seedlingscv. Pallas by means of soil drench at a final concentration of 8 mg/lsoil volume. The suspension used was made with tap water. Soil drenchwith the carrier material (wettable powder) acted as the control. Afterthree days, the plants were infected with Blumeria graminis (DC) Speerf.sp. hordei Em. Marchal, race A6 (5 conidia/mm²). Plants withchemically induced resistance (CIR) showed approximately 70% fewermildew colonies than the corresponding control plants which had onlybeen treated with the carrier substance (FIG. 9A).

To determine the amounts of BI1 transcripts, Northern blot and RT-PCTblots were carried out; they revealed a surprising reduction of theexpression of BI1 1 to 3 days after the chemical treatment (FIG. 9B).

Example 3 RNA Extraction

Total RNA was extracted from 8 to 10 primary leaf segments (length 5 cm)by means of “RNA extraction buffer” (AGS, Heidelberg, Germany). To thisend, the central primary leaf segments 5 cm in length were harvested andhomogenized in liquid nitrogen in mortars. The homogenate was stored at−70° C. until the RNA was extracted. Total RNA was extracted from thedeep-frozen leaf material with the aid of an RNA extraction kit (AGS,Heidelberg). To this end, 200 mg of the deep-frozen leaf material werecovered with 1.7 ml RNA extraction buffer (AGS) in a microcentrifugetube (2 ml) and immediately mixed thoroughly. After addition of 200 μlof chloroform, the mixture was again mixed thoroughly and shaken for 45minutes on a horizontal shaker at 200 rpm at room temperature. Toseparate the phases, the tubes were subsequently centrifuged for 15minutes at 20 000 g and 4° C., and the upper, aqueous phase wastransferred into a fresh microcentrifuge tube, while the bottom phasewas discarded. The aqueous phase was repurified with 900 μl ofchloroform by homogenizing for 10 seconds and recentrifuging (see above)and removing the aqueous phase (3 times). Then, 850 μl of 2-propanolwere added and the mixture was homogenized and placed on ice for 30 to60 minutes in order to precipitate the RNA. Thereafter, the mixture wascentrifuged for 20 minutes (see above), the supernatant was carefullydecanted off, 2 ml of 70% strength ethanol (−20° C.) were pipetted in,and the mixture was mixed and recentrifuged for 10 minutes. Then, thesupernatant was again decanted off, and the pellet was carefully freedfrom residual fluid, using a pipette, and then dried in a stream ofclean air on a clean bench. Then, the RNA was dissolved in 50 μl of DEPCwater on ice, mixed and centrifuged for 5 minutes (see above). 40 μl ofthe supernatant, constituting the RNA solution, were transferred into afresh microcentrifuge tube and stored at −70° C.

The RNA concentration was determined photometrically. To this end, theRNA solution was diluted 1:99 (v/v) with distilled water, and theabsorption was measured at 260 nm (Beckman Photometer DU 7400); (E₂₆₀nm=1 at 40 μg RNA/ml). The concentrations of the RNA solutions weresubsequently adjusted to 1 ∝g/∝l with DEPC water to match the calculatedRNA contents and verified in an agarose gel.

To verify the RNA concentrations in a horizontal agarose gel (1% agarosein 1×MOPS buffer with 0.2 μg/ml ethidium bromide), 1 μl of RNA solutionwas treated with 1 μl of 10×MOPS, 1 μl of color marker and 7 μl of DEPCwater, separated according to size in 1×MOPS running buffer over 1.5hours at a voltage of 120 V in the gel, and photographed under UV light.Any differences in concentration of the RNA extracts were adjusted withDEPC water, and the adjustment was rechecked in the gel.

Example 4 Cloning the BI1 cDNA Sequence from Barley

The full-length clone of hvBI1 (GenBank Acc.-No.: AJ290421) comprisestwo stop codons at the 3′ end and a potential start codon at the 5′ end.The ORF spans 247 amino acids and shows the highest degree of sequencehomology with a BI1 gene from rice, maize, Brassica napus andArabidopsis thaliana (in each case 86% identity at the nucleotide level)and a human BI1 homolog (53% similarity) (FIGS. 1 and 6). The amino acidsequence of hvBI1 comprises seven potential transmembrane domains withan orientation of the C terminus in the cytosol.

The following constructs were prepared:

a) Amplification of a 478 bp fragment of the barley BI1 cDNA (GenBankAcc.-No.: AJ290421) BI1-sense 5′-atggacgccttctactcgacctcg-3′BI1-antisense 5′- gccagagcaggatcgacgcc-3′

b) Amplification of a 513 bp Ubi cDNA fragment (GenBank Acc.-No.:M60175) UBI-sense 5′-ccaagatgcagatcttcgtga-3′ UBI-antisense5′-ttcgcgataggtaaaagagca-3′

c) Amplification of an 871 bp full-length BI1 reading frame BI1VL sense5′-ggattcaacgcgagcgcaggacaagc-3′ BI1VL antisense5′-gtcgacgcggtgacggtatctacatg-3′

The fragments obtained were ligated into the vector pGEM-T by means ofT-overhang ligation and acted as starting plasmids for the generation ofprobes (for example for Northern blot) or dsRNA. The individualconstructs were referred to as pGEMT-BI1, pGEMT-BI1VL(240) andpGEMT-UBI.

The BI1 full-length product was recloned from pGEMT into the SalIcleavage site of the pGY-1 vector (Schweizer, P., Pokorny, J.,Abderhalden, O. & Dudler, R. (1999) Mol. Plant-Microbe Interact. 12,647-654) using the SalI cleavage site in pGEMT and by means of the SalIcleavage sites which had been attached to the BI1VL antisense primer.Vectors with sense (pBI-1) and antisense (pasBI-1) orientation wereisolated and resequenced. The vectors comprise the BI-1 sequence underthe control of the CaMV 35S promoter.

Example 5 Reverse Transcription—Polymerase Chain Reaction (RT-PCR)

To detect small amounts of transcript, a semiquantiative RT-PCR wascarried out using the “OneStep RT-PCR Kit” (Qiagen, Hilden, Germany). Indoing so, RNA (isolated as above) was first translated into cDNA(reverse transcription) and the sought cDNA was amplified in asubsequent PCR reaction using specific primers. To estimate the initialamount of template RNA, the amplification was interrupted during theexponential phase (after 20 cycles) in order to reflect differences inthe target RNA. The PCR products were separated by means of an agarosegel, denatured, blotted onto nylon membranes, and detected with specificnon-radiolabeled probes under stringent standard conditions.Hybridization, wash steps and immunodetection were carried out asdescribed under “Northern blot”. The following components were combinedfor the individual reactions (25 μl batch) using the “One Step RT-PCRKit” (Qiagen, Hilden, Germany):

-   -   1000 ng total RNA of a specific sample    -   0.4 mM dNTPs    -   0.6 μM of each sense and antisense primer    -   0.10 μl RNase inhibitor    -   1 μl enzyme mix in 1×RT buffer.

cDNA synthesis (reverse transcription) was carried out for 30 minutes at50° C. The reverse transcriptase was subsequently inactivated for 15minutes at 95° C., which simultaneously causes activation of DNApolymerase and denaturation of cDNA. A PCR was subsequently carried outwith the following program: 1 minute at 94° C.; 25 cycles of 1 minute at94° C.; 1 minute at 54° C. and 1 minute at 72° C.; 10 minutes at 72° C.completion. Then storage at 4° C. until further use. The PCR productswere separated in a 1×TBE agarose gel using ethidium bromide. The aboveprimer pairs were used for the amplifications in the individual batches.

Example 6 Northern Blot Analysis

To prepare the Northern blotting, the RNA was separated in agarose gelunder denaturing conditions. To this end, part of the RNA solution(corresponding to 10 μg of RNA) was mixed with an identical volume ofsample buffer (with ethidium bromide), denatured for 5 minutes at 94°C., placed on ice for 5 minutes, centrifuged briefly and applied to thegel. The 1×MOPS gel (1.5% agarose, ultra pure grade) comprised 5 percentby volume of concentrated formaldehyde solution (36.5% [v/v]). The RNAwas separated for 2 hours at 100 V and subsequently blotted.

Northern blotting was done as an upward capillary RNA transfer. To thisend, the gel was first agitated gently for 30 minutes in 25 mM sodiumhydrogen/dihydrogen phosphate buffer (pH 6.5) and cut to size. A pieceof Whatman paper was prepared in such a way that it rested on ahorizontal slab and extended on 2 sides into a trough with 25 mM sodiumhydrogen/dihydrogen phosphate buffer (pH 6.5). This piece of paper wascovered with the gel, uncovered parts of the piece of Whatman paperbeing covered with a plastic film. The gel was then covered with apositively charged nylon membrane (Boehringer-Mannheim), avoiding airbubbles, whereupon the membrane was recovered to a height ofapproximately 5 cm with a stack of blotting paper. The blotting paperwas additionally weighed down with a sheet of glass and with a 100 gweight. Blotting was carried out overnight at room temperature. Themembrane was rinsed briefly in twice-distilled water and irradiated withUV light in a crosslinking apparatus (Biorad) with a light energy of 125mJ in order to immobilize the RNA. The uniformity of the RNA transfer tothe membrane was checked on a UV-light bench.

To detect barley mRNA, 10 mg of total RNA from each sample were resolvedin an agarose gel and blotted onto a positively charged nylon membraneby capillary transfer. Detection was effected using the DIG systemaccording to manufacturing specifications with digoxygenin-labeledantisense RNA probes (as described in Hückelhoven R et al. (2001) PlantMol Biol 47:739-748).

Probe preparation: Digoxygenin- or fluorescein-labeled RNA probes wereprepared for hybridization with the mRNAs to be detected. The probeswere generated by in-vitro transcription of a PCR product by means of aT7 or SP6 RNA polymerase, using labeled UTPs. The template for thePCR-aided amplification was provided by the above-described plasmidvectors pGEMT-BI1, pGEMT-UBI. Depending on the orientation of theinsert, different RNA polymerases were used for generating the antisensestrand. T7-RNA polymerase was used for pGEMT-BI1, while SP6—RNApolymerase was used for pGEMT-UBI. The insert of the individual vectorwas amplified via PCR using flanking standard primers (M13 fwd and rev).The reaction proceeded with the following end concentrations in a totalvolume of 50 μl of PCR buffer (Silverstar): M13-fwd:5′-GTAAAACGACGGCCAGTG-3′ M13-rev: 5′-GGAAACAGCTATGACCATG-3′

-   -   10% dimethyl sulfoxide (v/v)    -   2 ng/μl of each primer (M13 forward and reversed)    -   1.5 mM MgCl₂,    -   0.2 mM dNTPs,    -   4 units Taq polymerase (Silverstar),    -   2 ng/μl plasmid DNA.

The amplification was carried out in a Thermocycler (Perkin-Elmar 2400)with the following temperature program: 94° C. for 3 minutes; 30 cyclesof 30 seconds at 94° C.; 30 seconds at 58° C.; 1.2 minutes at 72° C.;72° C. for 5 minutes; then cooling at 4° C. until further use. Thesuccess of the reaction was verified in a 1% strength agarose gel. Theproducts were subsequently purified using a “High Pure PCR-ProductPurification Kit” (Boehringer-Mannheim). This gave approximately 40 μlof column eluate, which was again verified in the gel and stored at −20°C.

The RNA polymerization, the hybridization and the immuno-detection werecarried out largely following the kit manufacturer's instructionsregarding the nonradioactive RNA detection (DIG System User's Guide,DIG-Luminescence detection Kit, Boehringer-Mannheim, Kogel et al. (1994)Plant Physiol 106:1264-1277). 4 μl of purified PCR product were treatedwith 2 μl of transcription buffer, 2 μl of NTP labeling mix, 2 μl of NTPmix and 10 μl of DEPC water. Then, 2 μl of the T7 RNA polymerasesolution were pipetted in. The reaction was then carried out for 2 hoursat 37° C. and then made up to 100 μl with DEPC water. The RNA probe wasdetected in an ethidium bromide gel and stored at −20° C.

To prepare the hybridization, the membranes were first agitated gentlyfor 1 hour at 68° C. in 2×SSC (salt, sodium citrate), 0.1% SDS buffer(sodium dodecyl sulfate), the buffer being renewed twice or 3 times. Themembranes were subsequently applied to the internal wall ofhybridization tubes preheated at 68° C. and incubated for 30 minuteswith 10 ml of Dig-Easy hybridization buffer in a preheated hybridizationoven. In the meantime, 10 μl of probe solution were denatured for 5minutes at 94° C. in 80 μl of hybridization buffer, and the mixture wassubsequently placed on ice and centrifuged briefly. For thehybridization, the probe was then transferred into 10 ml ofhybridization buffer at a temperature of 68° C., and the buffer in thehybridization tube was replaced by this probe buffer. Hybridization wasthen carried out overnight, likewise at 68° C. Prior to theimmunodetection of RNA-RNA hybrids, the blots were washed twice understringent conditions for in each case 20 minutes in 0.1% (w/v) SDS,0.1×SSC at 68° C. For the immunodetection, the blots were first agitatedgently twice for 5 minutes in 2×SSC, 0.1% SDS at RT. 2 stringent washsteps were subsequently carried out for in each case 15 minutes at 68°C. in 0.1×SSC, 0.1% SDS. The solution was then replaced by wash bufferwithout Tween. The reaction mix was shaken for 1 minute and the solutionwas exchanged for blocking reagent. After a further 30 minutes' shaking,10 μl of antifluorescein antibody solution were added, and shaking wascontinued for 60 minutes. This was followed by two 15-minute wash stepsin Tween-containing wash buffer. The membrane was subsequentlyequilibrated for 2 minutes in substrate buffer and, after being left todrain, transferred to a sheet of acetate paper. A mixture of 20 μlCDP-Star™ and 2 ml of substrate buffer was then divided uniformly on the“RNA side” of the membrane. The membrane was subsequently covered with asecond sheet of acetate paper and the edges were heat-sealed to providea water-tight seal, avoiding air bubbles. In a dark room, the membranewas then covered for 10 minutes with an X-ray film and the film wassubsequently developed. The exposure time was varied as a function ofthe intensity of the luminescent reaction.

Unless otherwise specified, the solutions were part of the kit asdelivered (DIG-Luminescence detection Kit, Boehringer-Mannheim). All theothers were prepared from the following stock solutions by dilution withautoclaved distilled water. Unless otherwise specified, all the stocksolutions were made with DEPC (like DEPC water) and subsequentlyautoclaved.

-   -   DEPC water: distilled water is treated overnight at 37° C. with        diethyl pyrocarbonate (DEPC, 0.1%, w/v) and subsequently        autoclaved.    -   10×MOPS buffer: 0.2 M MOPS (morpholine-3-propanesulfonic acid),        0.05 M sodium acetate, 0.01 M EDTA, pH brought to 7.0 with 10 M        NaOH.    -   20×SSC (sodium chloride/sodium citrate, salt/sodium citrate): 3        M NaCl, 0.3 M trisodium citrate×2H₂O, pH brought to 7.0 with 4 M        HCl.    -   1% SDS (sodium dodecyl sulfate) sodium dodecyl sulfate (w/v),        without DEPC.    -   RNA sample buffer: 760 μl formamide, 260 μl formaldehyde, 100 μl        ethidium bromide (10 mg/ml), 80 μl glycerol, 80 μl bromophenol        blue (saturated), 160 μl 10×MOPS, 100 μL water.    -   10×wash buffer without Tween: 1.0 M maleic acid, 1.5 M NaCl;        without DEPC, bring to pH 7.5 with NaOH (solid, approx. 77 g)        and 10 M NaOH.    -   Tween-containing wash buffer: made by adding Tween to wash        buffer without Tween (0.3%, v/v).    -   10×blocking reagent: suspend 50 g of blocking powder        (Boehringer-Mannheim) in 500 ml of wash buffer without Tween.    -   Substrate buffer: bring 100 mM Tris        (trishydroxymethylaminomethane), 150 mM NaCl to pH 9.5 with 4 M        HCl.    -   10×color marker: 50% glycerol (v/v), 1.0 mM EDTA pH 8.0, 0.25%        bromophenol blue (w/v), 0.25% xylene cyanol (w/v).

A BI1 expression was analyzed as described using RT-PCR and cDNA gelblots and revealed that BI1 is predominantly expressed in the mesophylltissue of leaves, while ubiquitin is constitutively expressed uniformlyin epidermis and mesophyll (FIG. 8).

Expression of BI1 as response to treatment of the plants with toxicculture filtrates of Bipolaris sorokiniana can furthermore be observed.Barley primary leaves show typical necrotic lesions (leaf spot blotchsymptoms) after treatment of the plants with toxic culture filtrates ofBipolaris sorokiniana (procedure as described by Kumar et al. 2001). Theleaf necroses were discernible 48 hours post-treatment. The tissuedamage observed was more pronounced in the Bgh-resistant lineBcIngrid-mlo5 (122) than in the parent line Ingrid (Mlo genotype, Kumaret al. 2001). 72 hours post-treatment (hat), the expression of BI1correlates with the manifestation of the leaf necroses (FIG. 12).

Example 7

The wheat oxalate oxidase promoter (germin 9f-2.8) is employed to obtainstable, mesophyll-specific overexpression. In barley, the correspondingoxalate oxidase expression is mesophyll-specific, weakly constitutiveand pathogen-responsive (Gregersen P L et al. (1997) Physiol Mol PlantPathol 51: 85-97). It can therefore be utilized for themesophyll-specific expression of BI1. As a control, HvBI1 isoverexpressed under the control of the maize ubiquitin promoter(Christensen A H et al. (1992) Plant Mol Biol 18:675-689) or of the riceactin promoter (Zhang W et al. (1991) Plant Cell 3:1155-1165). Thefollowing constructs are employed:

-   a) pUbiBI-1 (SEQ ID NO: 33; for the transient transformation of    barley and wheat by means of particle bombardment. Expression of    BI-1 under the control of the maize ubiquitin promoter).-   b) pLo114UbiBI-1 (SEQ ID NO: 34; obtained by recloning the Ubi/BI-1    expression cassette as EcoR1 fragment from pUbiBI-1 in    pLo114-GUS-Kan; binary vector for the transient transformation of    barley with A. tumefaciens)-   c) pOXoBI-1 (SEQ ID NO: 35; mesophyll-specific TaGermin 9f-2.8    promoter upstream of BI1 for the transformation of wheat via    particle bombardment.-   d) pLo1140XoBI-1 (SEQ ID NO: 36)

Wild-type barley, wheat and mlo barley are transformed, propagated andselfed. The transformation of barley and wheat proceeds as described(Repellin A et al. (2001) Plant Cell, Tissue and Organ Culture 64:159-183): To this end, calli from immature wheat (or barley) embryos aretransformed via biolistic gene transfer with microprojectiles. pUC-basedvectors together with vectors which bear selection markers arecotransformed here. Thereafter, the embryos are grown on selectionmedium and regenerated. Barley is transformed with the aid ofAgrobacterium tumefaciens. A binary vector based on pCambia_(—)1301 isemployed for this purpose. Immature barley embryos are cocultured withA. tumefaciens, selected and subsequently regenerated (Repellin A et al.(2001) Plant Cell, Tissue and Organ Culture 64: 159-183; Horvath H etal. (2003) Proc Natl. Acad Sci USA 100: 365-369; Horvath H et al. (2002)in Barley Science, eds. Slafer, G. A., Molina-Cano, J. L., Savin, R.,Araus, J. L. & Romagosa, J. (Harworth, New York), pp. 143-176; Tingay Set al. (1997) Plant J. 11: 1369-1376).

The transgenic (recombinant) barley and wheat plants of the T1 or T2generation are studied for resistance to hemibiotrophic andperthotrophic pathogens. To this end, the leaves are inoculated with avariety of pathogens. The biotrophic pathogens used are powdery mildewof barley (Blumeria graminis f.sp. hordei) and leaf rust (Pucciniahordei). As a measure for the susceptibility to mildew, the number ofpustules per unit leaf area is evaluated 5-7 days after inoculation with2-5 conidia per mm² of leaf area (Beβer K et al. (2000) Mol PlantPathology 1: 277-286). Bipolaris sorokiniana and Magnaporthe grisea areused as hemibiotrophic pathogens. Inoculation is as described above(Kumar J et al. (2001) Phytopathology 91: 127-133; Jarosch B et al.(1999) Mol Plant Microbe Inter 12: 508-514). The number and size of theleaf lesions 2 to 6 days after spray inoculation with conidia is used asmeasure for the susceptibility (Kumar J et al. (2001) Phytopathology91:127-133; Jarosch B et al. (1999) Mol Plant Microbe Inter 12:508-514;Jarosch B et al. (2003) Mol Plant Microbe Inter 16:107-114.). Fusariumgraminearum is used as perthotrophic pathogen.

To determine the fusarium head blight (FHB) type-I resistance, wheatears in an early stage of flowering are sprayed with a macroconidialsuspension (approx. 2×10⁵ ml⁻¹) of Fusarium graminearum and of Fusariumculmorum, respectively. The inoculated plants are transferred for 3 daysinto a humid chamber with an air temperature of 25° C. and a relativeatmospheric humidity of 100%. Thereafter, the plants are incubated inthe greenhouse under continuous light at a temperature of 20° C., andthe severity of the FHB symptoms along the ear are evaluated after 5, 7and 8 days.

To quantify the fusarium head blight (FBH) type-II resistance, in eachcase 10-20 μl aliquots of a macroconidial suspension (approx. 2×10⁵ml⁻¹) of Fusarium graminearum and Fusarium culmorum, respectively, areinjected into individual, relatively centrally located spikelets ofwheat plants. The inoculated plants are transferred for 3 days into ahumid chamber at an air temperature of 25° C. and a relative atmospherichumidity of 100%. Thereafter, the plants are incubated in the greenhouseunder continuous light at a temperature of 20° C., and the spreading ofthe FHB symptoms along the ear is evaluated after 7, 14 and 21 days. Thespreading of the symptoms along the ear (what is known as Fusariumspreading) is taken as a measure for the FHB type-II resistance.

Comparative Example 1 Transient BI1 Expression in the Epidermis, andEvaluation of the Development of the Fungal Pathogen

Barley cv Ingrid leaf segments were transformed with a pGY-BI1 togetherwith a GFP expression vector. Thereafter, the leaves were inoculatedwith Bgh, and the result was analyzed after 48 hours by means of lightand fluorescent microscopy. The penetration in GFP-expressing cells wasassessed by detecting haustoria in live cells and by assessing thefungal development in precisely those cells. A transient transformationmethod which had already been described for the biolistic introductionof DNA and RNA into epidermal cells of barley leaves was employed(Schweizer P et al. (1999) Mol Plant Microbe Interact 12:647-54;Schweizer P et al. (2000) Plant J 2000 24:895-903).

To prepare the microcarriers, 55 mg of tungsten particles (M 17,diameter 1.1 μm; Bio-Rad, Munich) were washed twice with 1 ml ofautoclaved distilled water and once with 1 ml of absolute ethanol, driedand taken up in 1 ml of 50% strength glycerol (approx. 50 mg/ml stocksolution). The solution was diluted to 25 mg/ml with 50% strengthglycerol, mixed thoroughly prior to use, and suspended in an ultrasonicbath.

To coat the microcarriers for each bombardment, 0.3 μg of plasmid pGFP(GFP under the control of the CaMV 35S promoter; Schweizer P et al.(1999) Mol Plant-Microbe Interact 12:647-654.), 0.7 μg of blank vectorpGY or pGY-BI1 (1 μl), 12.5 μl of tungsten particle suspension (25mg/ml; corresponding to 312 μg of tungsten particles), 12.5 μl of 1 MCa(NO₃)₂ solution (pH 10) were combined dropwise with constant mixing,the mixture was left to stand for 10 minutes at RT and then brieflycentrifuged, and 20 μl of the supernatant were drawn off. The remainderwith the tungsten particles is resuspended (ultrasonic bath) andemployed in the experiment.

Segments (approx. 4 cm in length) of barley primary leaves were used.The tissue was placed on 0.5% Phytagar (GibcoBRL™ Life Technologies™,Karlsruhe) supplemented with 20 μg/ml benzimidazole in Petri dishes(diameter 6.5 cm), and the edges were covered directly prior to particlebombardment with a stencil provided with a rectangular opening of 2.2cm×2.3 cm. One after the other, the dishes were placed on the bottom ofthe vacuum chamber (Schweizer P et al. (1999) Mol Plant Microbe Interact12:647-54) over which a nylon mesh (mesh size 0.2 mm, Millipore,Eschborn) on an apertured plate had been inserted (5 cm above thebottom, 11 cm underneath the macrocarrier, see hereinbelow) to act asdiffuser in order to disperse particle aggregates and to slow down theparticle stream. For each bombardment, the macrocarrier (plastic sterilefilter holder, 13 mm, Gelman Sciences, Swinney, UK), which was attachedat the top of the chamber, was loaded with 5.8 μl of DNA-coated tungstenparticles (microcarrier, see hereinbelow). The pressure in the chamberwas reduced by 0.9 bar using a diaphragm vacuum pump (Vacuubrand,Wertheim), and the surface of the plant tissue was bombarded with thetungsten particles at a helium gas pressure of 9 bar. The chamber wasaerated immediately thereafter. To label transformed cells, the leaveswere bombarded with the plasmid (pGFP; vector pUC18-based, CaMV 35Spromoter/terminator cassette with inserted GFP gene; Schweizer P et al.(1999) Mol Plant Microbe Interact 12:647-54; provided by Dr. P.Schweizer, Institut für Pflanzengenetik [Department of Plant Genetics]IPK, Gatersleben, Germany). Each time before another plasmid was usedfor the bombardment, the macrocarrier was cleaned thoroughly with water.Following incubation for four hours after the bombardment with slightlyopen Petri dishes, RT and daylight, the leaves were inoculated with 100conidia/mm² of the barley powdery mildew fungus (race A6; Blumeriagraminis f. sp. hordei mildew A6) and incubated for a further 40 hoursunder identical conditions. The penetration was then evaluated. Theresult (for example the penetration efficiency), defined as percentageof attacked cells with a mature haustorium and a secondary hypha(secondary elongating hyphae) was analyzed by fluorescence and lightmicroscopy. Inoculation with 150 conidia/mm² results in an attackfrequency of approximately 50% of the transformed cells. A minimum of100 interaction sites were evaluated for each individual experiment.Transformed (GFP-expressing) cells were identified under excitation withblue light. Three different categories of transformed cells weredistinguished:

-   1. Penetrated cells comprising a readily recognizable haustorium. A    cell with more than one haustorium counted as one cell.-   2. Cells which were attacked by a fungal appressorium, but comprise    no haustorium. A cell which was attacked repeatedly by Bgh, but    comprises no haustorium, counted as one cell.-   3. Cells which are rot attacked by Bgh.

Stomatal cells and subsidiary stomatal cells were excluded from theevaluation. Surface structures of Bgh were analyzed by light microscopyor fluorescent staining of the fungus with 0.3% Calcofluor (w/v inwater) for 30 sec. Fungal development can be evaluated readily bystaining with Calcofluor followed by fluorescence microscopy. While thefungus develops a primary germ tube and an appressorial germ tube incells transformed with BI1-dsRNA, it fails to develop a haustorium. Thedevelopment of haustoria is a precondition for the formation of asecondary hypha.

-   -   The penetration efficiencies (penetration rates) are calculated        as the number of penetrated cells divided by the number of the        attacked cells, multiplied by 100.

The penetration efficiency is used for determining the susceptibility ofcells which are transfected with pGY-BI1 in comparison with cells whichare transformed with a blank vector control (FIG. 10). It can be seenthat the overexpression of BI1 significantly increases the penetrationfrequency of Bgh (FIG. 10). In six independent experiments,overexpression in the susceptible barley variety Ingrid brought about asignificant increase in the average penetration efficiency (PE) from 47%to 72% (165% of the controls) in cells which overexpress BI1 incomparison with cells which were transformed with blank vector (control)(FIG. 10).

Furthermore, epidermal cells of the Bgh-resistant m1o5 barley weretransformed transiently as described above with the BI1 overexpressionconstruct pGY-1. The mlo5 genotype in a Pallas or Ingrid backgroundshows minor susceptibility to Bgh. In 7 independent experiments, apenetration efficiency of a minimum of 0 to a maximum of 11% was foundin control plants (transformation with blank vector and GFP vector).Surprisingly, BI1 overexpression (pGY-BI1) resulted in a virtuallycomplete reconstitution of the susceptible phenotype, i.e. the mloresistance was broken almost completely. The average penetrationefficiency of Bgh on Ingrid-mlo5 and Pallas-mlo5 leaf segments climbsfrom 4% to 23% and from 6% to 33%, respectively (FIG. 11). This means arelative increase in penetration to 520% and 510%, respectively, of thecontrols. Moreover, the overexpression of BI1 in barley cv Manchuriaincreased the susceptibility to the wheat pathogen Blumeria graminisf.sp. tritici from 0 to 4% to 19 to 27% in three independent experiments(FIG. 13).

1. A method for generating or increasing the resistance, in plants, toat least one biotic or abiotic stress factor, comprising the followingsteps: a) increasing the amount of protein, or the function, of at leastone Bax inhibitor-1 (BI1) protein in at least one plant tissue with theproviso that the expression in the leaf epidermis remains essentiallyunchanged or is reduced, and b) selection of the plants in which, incomparison with the starting plant, a resistance to at least one bioticor abiotic stress factor exists or is increased.
 2. The method accordingto claim 1, wherein the stress factor is a plant pathogen.
 3. The methodaccording to claim 1, wherein the stress factor is a necrotrophic orhemibiotrophic pathogen.
 4. The method according to claim 1, wherein theBI-1 protein comprises at least one sequence which has at least 50%homology with at least one BI1 consensus motif selected from the groupconsisting of a) H(L/I)KXVY (SEQ ID NO: 45) b) AXGA(Y/F)XH (SEQ ID NO:46) c) NIGG (SEQ ID NO: 47) d) P(V/P)(Y/F)E(E/Q)(R/Q)KR (SEQ ID NO: 48)e) (E/Q)G(A/S)S(V/I)GPL (SEQ ID NO: 49) f) DP(S/G)(L/I)(I/L) (SEQ ID NO:50) g) V(G/A)T(A/S)(L/I)AF(A/G)CF(S/T) (SEQ ID NO: 51) h) YL(Y/F)LGG,(SEQ ID NO: 52) preferably EYLYLGG (SEQ ID NO: 53) i)L(L/V)SS(G/W)L(S/T)(I/M)L(L/M)W (SEQ ID NO: 54) j) DTGX(I/V)(I/V)E. (SEQID NO: 55)


5. The method according to claim 1, wherein the BI-1 protein is encodedby a polypeptide comprising at least one sequence selected from thegroup consisting of: a) the sequences as shown in SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 and 38, and b) sequenceswhich have at least 50% identity with one of the sequences as shown inSEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32and 38, c) sequences which comprise at least one part-sequence of atleast 10 contiguous amino acid residues of one of the sequences as shownin SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32and
 38. 6. The method according to claim 1, wherein the increase in theprotein quantity or function of at least one BI1 protein is effected byrecombinant expression of said BI1 protein under the control of a root-,tuber- or mesophyll-specific promoter.
 7. The method according to claim1, comprising (a) stably transforming a plant cell with a recombinantexpression cassette comprising a nucleic acid sequence coding for a BIprotein in functional linkage with a tissue-specific promoter, thepromoter having essentially no activity in the leaf epidermis and thepromoter being heterologous with regard to said nucleic acid sequencewhich codes for the BI protein; (b) regenerating the plant from theplant cell; and (c) expressing said nucleic acid sequence which codesfor a BI protein in an amount and for a period sufficient to generate orto increase a stress and/or pathogen resistance in said plant.
 8. Themethod according to claim 1, wherein the plant is selected from amongthe monocotyledonous and dicotyledonous plants.
 9. The method accordingto claim 1, wherein the plant is selected from the group of themonocotyledonous plants consisting of wheat, oats, millet, barley, rye,maize, rice, buckwheat, sorghum, triticale, spelt, linseed and sugarcane.
 10. The method according to claim 1, wherein the expression of theBax inhibitor-1 (BI-1) in the mesophyll is increased.
 11. The methodaccording to claim 1, wherein the plant has an mlo-resistant phenotype,or the expression or function of MLO, RacB and/or NaOx is inhibited or,in comparison with a control plant, is reduced at least in the epidermisand/or the expression or function of PEN2, SNAP34 and/or PEN1 isincreased at least in the epidermis in comparison with a control plant.12. A polypeptide sequence coding for a BI1 protein comprising at leastone sequence selected from the group consisting of a) the sequences asshown in SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 28, 30, 32 or 38, b)sequences which have at least 90%, preferably at least 95%, especiallypreferably at least 98%, homology with one of the sequences as shown inSEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 28, 30, 32 or 38, and c)sequences which comprise at least 10, preferably at least 20, especiallypreferably at least 30, contiguous amino acids of one of the sequencesas shown in SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 28, 30, 32 or
 38. 13.A nucleic acid sequence coding for a polypeptide sequence according toclaim
 12. 14. A recombinant expression cassette comprising a nucleicacid sequence coding for a BI protein in functional linkage with atissue-specific promoter, the promoter having essentially no activity inthe leaf epidermis and the promoter being heterologous with regard tosaid nucleic acid sequence which codes for the BI protein.
 15. Therecombinant expression cassette comprising a nucleic acid sequencecoding for a BI protein in functional linkage with a tissue-specificpromoter, the promoter having essentially no activity in the leafepidermis and the promoter being heterologous with regard to saidnucleic acid sequence which codes for the BI protein, where a) the BI1protein is as defined in claim 4, and/or b) the tissue-specific promoteris selected from the group of the root-, tuber- or mesophyll-specificpromoters.
 16. A recombinant vector comprising an expression cassetteaccording to claim
 14. 17. A recombinant organism comprising at leastone expression cassette according to claim
 14. 18. The recombinantorganism according to claim 17 selected from the group consisting ofbacteria, yeasts, nonhuman animals and plants.
 19. The recombinantorganism according to claim 17 or 18, selected from the group of theplants consisting of wheat, oats, millet, barley, rye, maize, rice,buckwheat, sorghum, triticale, spelt, linseed, sugar cane, oilseed rape,cress, Arabidopsis, cabbage species, soybean, alfalfa, pea, beans,peanut, potato, tobacco, tomato, eggplant, paprika, sunflower, Tagetes,lettuce, Calendula, melon, pumpkin/squash and zucchini.
 20. Therecombinant organism according to claim 17, wherein the organism is aplant which additionally has an mlo-resistant phenotype.
 21. Therecombinant expression cassette comprising a nucleic acid sequencecoding for a BI protein in functional linkage with a tissue-specificpromoter, the promoter having essentially no activity in the leafepidermis and the promoter being heterologous with regard to saidnucleic acid sequence which codes for the BI protein, where a) the BI1protein is as defined in claim 5, and/or b) the tissue-specific promoteris selected from the group of the root-, tuber- or mesophyll-specificpromoters.